Single-stranded rnai agents containing an internal, non-nucleic acid spacer

ABSTRACT

The instant disclosure features single-stranded RNA molecules comprising one or more internal, non-nucleotide spacers. A non-nucleotide spacer covalently links nucleotide portions of the molecule. The single-stranded RNA molecules function as guide or antisense strands that are capable of inhibiting gene expression via an RNA interference mechanism and, thus, represent single-stranded RNAi agents. The single-stranded RNAi molecules can be used in methods for a variety of therapeutic, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/967,974, filed Dec. 14, 2015, which is a continuation of U.S. patentapplication Ser. No. 13/818,306, filed Aug. 19, 2011, now U.S. Pat. No.9,243,246, issued Jan. 26, 2016, which is a National Stage Entry of PCTApplication No. PCT/US2011/048338, filed Aug. 19, 2011 which claims thebenefit of U.S. Provisional Application No. 61/376,471, filed Aug. 24,2010. Each of these prior applications is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

The RNA interference (RNAi) pathway is an evolutionarily conserved modeof gene regulation. The RNAi process is initiated by double-stranded RNA(dsRNA) produced from various exogenous or endogenous sources (e.g.,experimental introduction, viral infection). The dsRNA is cleaved byDicer to generate 20-25 nucleotide small-interfering RNA (siRNA)duplexes. These duplexes are then loaded onto the RNA-induced silencingcomplex (RISC), and before RISC is activated, the passenger/sense strandof the duplex is removed. The single guide/antisense strand remainsassociated with RISC and directs cleavage of the target mRNA. Thus,duplexed siRNA have become an important tool for both research andnucleic acid-based therapeutics.

RNAi gene silencing can occur via single-stranded or double-stranded RNAmolecules. In the last ten years, it has been reported thatsingle-stranded antisense siRNA are almost as potent as the siRNA duplex(see, e.g., Schwarz et al., 2002, Mol. Cell. 10:537-548; Martinez etal., 2002, Cell 110:563-574; Amarzguioui et al., 2003, Nucleic AcidsRes. 31:589-595; and Holen et al., 2003, Nucleic Acids Res.31:2401-2407). There are benefits in utilizing single-stranded RNAmolecules, as opposed to duplexed versions, for gene silencing. Theirlower molecule weight may make them easier to cross cell membranes.Single-stranded RNA molecules are also half the mass and volume ofduplexed siRNA, implicating a manufacturing cost advantage. Thus, thereremains a heightened interest in formulating new and advantageous designfeatures suitable for single-stranded RNAi molecules.

SUMMARY OF THE INVENTION

The instant disclosure provides single-stranded RNA molecules whichcomprise: (a) a nucleic acid portion comprising two or more nucleotideportions, and (b) an internal (as opposed to “terminal”) spacer portioncomprising one or more non-nucleotide spacer portions, wherein anon-nucleotide spacer portion covalently links two nucleotide portionsof the molecule. The nucleotide portions of the single-stranded RNAmolecules of the invention are not complementary to each other and,thus, said portions do not form base pairs. The single-stranded RNAmolecules of the invention function as guide or antisense strands thatare capable of inhibiting gene expression via an RNA interferencemechanism and, thus, represent single-stranded RNAi agents.

A single-stranded RNAi molecule of the invention has a single-strandedoligonucleotide structure and mediates RNA interference against a targetRNA. A single-stranded RNAi molecule comprises: (a) a nucleic acidportion comprising a first nucleotide portion (N1) and a secondnucleotide portion (N2), wherein said nucleic acid portion comprises atleast 8 nucleotides that can base pair with a target RNA, and whereinthe total number of nucleotides within the nucleic acid portion is from8 to 26 nucleotides; and, (b) an internal spacer portion comprising atleast a first non-nucleotide spacer portion (S1) that covalently linksthe first and second nucleotide portions. The first and secondnucleotide portions are not self complementary. The total number ofnucleotides of a single-stranded RNAi molecule of the invention (e.g., 8to 26) is distributed between the nucleotide portions of the molecule,wherein each nucleotide portion contains at least one nucleotide.

In one embodiment, the nucleic acid portion of a single-stranded RNAimolecule of the invention contains two nucleotide portions, referred toas the first nucleotide portion (N1) and the second nucleotide portion(N2). The first and second nucleotide portions of an RNAi molecule ofthe invention are covalently attached to a non-nucleotide spacer portionof the molecule. In another embodiment, the nucleic acid portion of asingle-stranded RNAi molecule of the invention contains more than onenucleotide portion (e.g., 3, 4, or 5, referred to as third (N3), fourth(N4) or fifth (N5) nucleotide portions, respectively).

In one embodiment, the internal spacer portion of a single-stranded RNAimolecule of the invention contains only one non-nucleotide spacerportion, referred to as the first non-nucleotide spacer portion (S1).The first non-nucleotide spacer portion (S1) of an RNAi molecule of theinvention is covalently attached to two nucleotides and/ornon-nucleotide substitutes, each located within a distinct nucleotideportion of the single-stranded molecule. In another embodiment, theinternal spacer portion of a single-stranded RNAi molecule of theinvention contains more than one non-nucleotide spacer portion (e.g., 2,3, or 4, referred to as second (S2), third (S3) or fourth (S4)non-nucleotide spacer portions, respectively).

A single-stranded RNAi molecule of the invention comprises a nucleotidesequence that is partially, substantially or perfectly complementary toan RNA target site in a cell.

In one embodiment, a single-stranded RNAi molecule of the inventioncomprises a nucleotide sequence that is partially, substantially, orperfectly homologous to the guide strand of a naturally-occurring miRNAand, thus, functions as a miRNA mimetic. A single-stranded miRNA mimeticof the invention is designed based on a corresponding,naturally-occurring miRNA, wherein at least one non-nucleotide spacerportion is either located between two adjacent nucleotides of thenaturally-occurring miRNA guide strand sequence or substituted for fromone to about 12 internal (i.e., non-terminal) nucleotides of thenaturally-occurring miRNA guide strand sequence.

In another embodiment, a single-stranded RNAi molecule of the inventionis an analog of either a single-stranded siRNA or the guide/antisensestrand of a duplex siRNA, wherein the single-stranded RNAi moleculecomprises a sequence that is partially, substantially, or perfectlyhomologous to the corresponding single-stranded siRNA or the guidestrand of the corresponding duplex siRNA. The correspondingsingle-stranded siRNA or duplex siRNA may be known to inhibit geneexpression via an RNAi mechanism. In this embodiment, thesingle-stranded RNAi molecule represents a single-stranded siRNAmimetic. A single-stranded siRNA mimetic of the invention is designedbased on a corresponding siRNA, wherein at least one non-nucleotidespacer portion is either located between two adjacent nucleotides of thesiRNA guide strand sequence or substituted for from one to about 4nucleotides of the corresponding siRNA guide strand sequence.

A single-stranded RNAi molecule of the invention can comprisesubstitutions, chemically-modified nucleotides, and non-nucleotides,including substitutions or modifications in the backbone, sugars, bases,or nucleosides. In certain embodiments, the use of substituted ormodified single-stranded RNAi molecules of this disclosure can enableachievement of a given therapeutic effect at a lower dose since thesemolecules may be designed to have an increased half-life in a subject orbiological samples (e.g., serum). Furthermore, certain substitutions ormodifications can be used to improve the bioavailability ofsingle-stranded RNAi molecules by targeting particular cells or tissuesor improving cellular uptake of the single-stranded RNAi molecules.

The internal spacer portion of a single-stranded RNAi molecule of theinvention can comprise one or more non-nucleotide spacer portions. Anon-nucleotide spacer portion can include any aliphatic or aromaticchemical group that can be further substituted, wherein said spacerportion does not contain a nucleotide. The spacer portion can besubstituted with a chemical moiety that provides additionalfunctionality to a single-stranded RNAi molecule. For example, anon-nucleotide spacer portion can be substituted with a moiety thatbinds specifically to a target molecule of interest orfacilitates/enhances cellular delivery of the molecule. In oneembodiment of the invention, a non-nucleotide spacer portion includes analkyl, alkenyl or alkynyl chain of preferably 1 to 20 carbons that canbe optionally substituted.

The single-stranded RNAi molecules of the invention are useful reagents,which can be used in methods for a variety of therapeutic, diagnostic,target validation, genomic discovery, genetic engineering, andpharmacogenomic applications. Thus, the prevent invention furtherincludes compositions comprising a single-stranded RNAi molecule of thedisclosure and methods for inhibiting expression of one or morecorresponding target mRNAs in a cell or organism. This disclosureprovides methods and single-stranded RNAi molecule compositions fortreating a subject, including a human cell, tissue or individual.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the degree of inhibition of VAMP3 target expression bysingle-stranded miR-124 analogs containing a C3 spacer using a RT-qPCRassay. The structure and sequence of the analogs are specificallydescribed in Table 2, infra. In the schematic drawings of the miR-124analogs, the circles represent nucleotides, with the exception of theblack circle located at the 5′ terminus which represents a 5′ phosphate.The open circles represent 2′-deoxy-2′-fluoro nucleotides. The blackcircles located at the 3′ terminus of the schematics represent2′-O-methyl nucleotides, and the “3” represents the location of the C3spacer. In the schematic of the “124(21)-8p-16rrr” analog, the threeinternal black circles represent unmodified ribonucleotides. The longerbars in the graph indicate greater knockdown, and duplicate barsindicate biological replicates.

FIG. 2 shows the degree of inhibition of VAMP3 target expression bysingle-stranded miR-124 analogs containing a C6 spacer using a RT-qPCRassay. The structure and sequence of the analogs are specificallydescribed in Table 2, infra. In the schematic drawings of the miR-124analogs, the circles represent nucleotides, with the exception of theblack circle located at the 5′ terminus which represents a 5′ phosphate.The open circles represent 2′-deoxy-2′-fluoro nucleotides. The blackcircles located at the 3′ terminus of the schematics represent2′-O-methyl nucleotides, and the “6” represents the location of the C6spacer. In the schematic of the “124(21)-8p-16rrr” analog, the threeinternal black circles represent unmodified ribonucleotides. The longerbars in the graph indicate greater knockdown, and duplicate barsindicate biological replicates.

FIG. 3 shows the dose-dependent response of VAMP3 expression for asubset of the analogs tested in FIGS. 1 and 2. VAMP3 expression isdepicted along the y-axis. The dose of the miR-124 analog tested (seeTable 2, infra, for structure and sequences) is depicted along thex-axis, ranging from the lowest doses on the left to the highest doseson the right.

FIG. 4 shows the degree of inhibition by single-stranded miR-124 analogscontaining a C3 spacer using a reporter assay that measures knockdown ofa co-transfected luciferase reporter that carries two target sitesmatching the seed region of miR-124. The structure and sequence of theanalogs are specifically described in Table 2, infra. In the schematicdrawings of the miR-124 analogs, the circles represent nucleotides, withthe exception of the black circle located at the 5′ terminus whichrepresents a 5′ phosphate. The open circles represent 2′-deoxy-2′-fluoronucleotides. The black circles located at the 3′ terminus of theschematics represent 2′-O-methyl nucleotides, and the “3” represents thelocation of the C3 spacer. In the schematic of the “124(21)-8p-16rrr”analog, the three internal black circles represent unmodifiedribonucleotides. The duplicate bars of the graph indicate biologicalreplicates, and longer bars indicate greater inhibition.

FIG. 5 shows the degree of inhibition by single-stranded miR-124 analogscontaining a C6 spacer using a reporter assay that measures knockdown ofa co-transfected luciferase reporter that carries two target sitesmatching the seed region of miR-124. The structure and sequence of theanalogs are specifically described in Table 2, infra. In the schematicdrawings of the miR-124 analogs, the circles represent nucleotides, withthe exception of the black circle located at the 5′ terminus whichrepresents a 5′ phosphate. The open circles represent 2′-deoxy-2′-fluoronucleotides. The black circles located at the 3′ terminus of theschematics represent 2′-O-methyl nucleotides, and the “6” represents thelocation of the C6 spacer. In the schematic of the “124(21)-8p-16rrr”analog, the three internal black circles represent unmodifiedribonucleotides. The duplicate bars indicate biological replicates, andlonger bars indicate greater inhibition.

FIGS. 6A and 6B show the dose-dependent response of target expressioninhibition of two different luciferase reporters for a subset of thesingle-stranded miR-124 analogs tested in FIG. 3. In FIG. 6A, theinhibition activity shown is against a luciferase reporter with twomatches to the miR-124 seed region, representing the miRNA activity ofthe tested analogs. In FIG. 6B, the inhibition activity shown is againsta luciferase reporter with two full-length matches to miR-124,representing the siRNA activity of the tested analogs.

FIG. 7 compares the knockdown of ApoB mRNA using ApoB-targeted singlestranded (guide strand) oligonucleotides having a C3 spacer incorporatedat either position 15 (“485 c3@pos15”), 16 (“485 c3@pos16”), 17 (“485c3@pos17”), 18 (“485 c3@pos185”), or 19 (“485 c3@pos19”) (relative tothe 5′ of the oligo) to the corresponding single strandedoligonucleotide without the spacer (“485”) at two differentconcentrations (100 nM and 10 nM). All of the single stranded moleculesare composed of 2′-deoxy-2′-fluoro nucleotides at both pyrimidine andpurine nucleotides, a 5′ phosphate, and two 2′-O-methyl nucleotides atthe 3′ terminus.

FIGS. 8 and 9 compares ApoB mRNA knockdown using 30 different singlestrand sequences targeting ApoB with C3 spacer at either position 18(FIG. 8) or position 19 (FIG. 9) at two concentrations (100 nM and 10nM). Single strands are notated on the x-axis by the position within theApoB mRNA which they target. All of the single stranded molecules arecomposed of 2′-deoxy-2′-fluoro nucleotides at both pyrimidine and purinenucleotides, a 5′ phosphate, and two 2′-O-methyl nucleotides at the 3′terminus.

FIG. 10 compares ApoB mRNA knockdown at 100 nM concentration usingsingle stranded molecules targeting each of the 30 different ApoB targetsites tested in FIGS. 8 and 9—single strands without a C3 spacer(“all-flu-p”), with a C3 spacer at position 18 (“all-flu-c3-18-p”), andwith a C3 spacer at position 19 (“all-flu-c3-19-p”). All of the singlestranded molecules are composed of 2′-deoxy-2′-fluoro nucleotides atboth pyrimidine and purine nucleotides, a 5′ phosphate, and two2′-O-methyl nucleotides at the 3′ terminus.

DETAILED DESCRIPTION OF THE INVENTION A. Terms and Definitions

The following terminology and definitions apply as used in the presentapplication.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a cell”includes a combination of two or more cells, and the like.

Any concentration range, percentage range, ratio range, or integer rangeis to be understood to include the value of any integer within therecited range, and when appropriate, fractions thereof (such as onetenth and one hundredth of an integer), unless otherwise indicated.

“About” or “approximately,” as used herein, in reference to a number aregenerally taken to include numbers that fall within a range of 5% ineither direction (greater than or less than) of the number unlessotherwise stated or otherwise evident from the context (except wheresuch number would exceed 100% of a possible value). Where ranges arestated, the endpoints are included within the range unless otherwisestated or otherwise evident from the context.

As used herein, the terms “including” (and any form thereof, such as“includes” and “include), “comprising” (and any form thereof, such as“comprise” and “comprises”), “having” (and any form thereof, such as“has” or “have”), or “containing” (and any form thereof, such as“contains” or “contain”) are inclusive and open-ended and do not excludeadditional, un-recited elements or method steps.

“Analog” as used herein refers to its meaning as is generally acceptedin the art. The term generally refers to a compound or molecule that isstructurally similar to a parent compound or molecule (e.g., anucleotide, a naturally-occurring miRNA), but differs slightly incomposition (e.g., one atom or functional group is different, added, orremoved). The analog may or may not have different chemical or physicalproperties than the original parent compound or molecule and may or maynot have improved biological or chemical activity. For example, theanalog may be more hydrophilic or it may have altered activity of theparent compound/molecule. The analog may be a naturally or non-naturallyoccurring (e.g., chemically-modified or recombinant) variant of theoriginal parent compound/molecule. An example of an RNA analog is an RNAmolecule comprising a nucleotide analog. A nucleotide analog is anucleotide that is chemically-modified at the sugar, base or nucleoside,as is generally recognized in the art.

As used herein, the term “mimetic” refers to its meaning as is generallyaccepted in the art. The term generally refers to a molecule that isstructurally different from a reference molecule. For example, areference molecule for purposes of certain embodiments of the presentinvention can be a naturally-occurring miRNA molecule, or asingle-stranded siRNA molecule, that does not contain a non-nucleotideinternal spacer. The mimetic is capable of performing one or more or allof the biological, physiological, and/or chemical functions that arewithin the capabilities of the reference molecule. The mimetic and thereference molecule do not have to be functional equivalents, but themimetic should be able to perform one or more functions and exhibit atleast 5% or more, 10% or more, 20% or more, 30% or more, 40% or more,50% or more, 60% or more, 70% or more, 80% or more, or 90% or more ofthe activity of the reference molecule, as measured and compared usingassays or parameters that are suitable to represent the sharedfunction(s). The terms “analog” and “mimetic,” when describing an RNAimolecule of the disclosure that is structurally different from areference RNAi molecule, can be used interchangeably.

The term “nucleotide” refers to its meaning as is generally recognizedin the art. Nucleotides generally comprise a nucleobase, a sugar, and aninternucleoside linkage, e.g., a phosphate. The base can be a naturalbase (standard), a modified base, or a base analog, as are well known inthe art. Such bases are generally located at the 1′ position of anucleotide sugar moiety. Additionally, the nucleotides can be unmodifiedor modified at the sugar, internucleoside linkage, and/or base moiety(also referred to interchangeably as nucleotide analogs, modifiednucleotides, non-natural nucleotides, and non-standard nucleotides);see, for example, U.S. application Ser. No. 12/064,014.

The terms “polynucleotide” and “oligonucleotide” as used herein refer tothe meaning as is generally accepted in the art. The terms generallyrefer to a chain of nucleotides. “Nucleic acids” and “nucleic acidmolecules” are polymers of nucleotides. Thus, “nucleic acids,”“polynucleotides” and “oligonucleotides” are interchangeable herein. Oneskilled in the art has the general knowledge that nucleic acids arepolynucleotides which can be hydrolyzed into monomeric nucleotides.Monomeric nucleotides can be further hydrolyzed into nucleosides.

By “a contiguous stretch of nucleotides” is meant a continuous series ofat least 2 nucleotides. The bonds connecting the nucleotides within thestretch are phosphodiester bonds.

The term “RNA” as used herein refers to its meaning as is generallyaccepted in the art. The term generally refers to a molecule comprisingat least one ribofuranoside residue, such as a ribonucleotide. The term“ribonucleotide” means a nucleotide with a hydroxyl group at the 2′position of a β-D-ribofuranose moiety. The term refers to adouble-stranded RNA, a single-stranded RNA, an isolated RNA such as apartially purified RNA, an essentially pure RNA, a synthetic RNA, arecombinantly-produced RNA, or an altered RNA that differs from anaturally-occurring RNA by the addition, deletion, substitution and/oralteration of one or more nucleotides therein. Such alterations caninclude addition of non-nucleotide material, for example, at one or morenon-terminal nucleotides of an RNA molecule. As such, nucleotides in thesingle-stranded RNA molecules of the invention can comprise non-standardnucleotides, such as non-naturally occurring nucleotides,chemically-synthesized and/or modified nucleotides, or deoxynucleotides.The altered RNA is referred to as a “modified RNA” or a “RNA analog.”

The term “pyrimidine” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to conventionalpyrimidine bases, including the standard pyrimidine bases uracil,thymidine, and cytosine. In addition, the term pyrimidine iscontemplated to embrace non-standard pyrimidine bases or acids, such as5-methyluracil, 2-thio-5-methyluracil, 4-thiouracil, pseudouracil,dihydrouracil, orotate, 5-methylcytosine, or the like, as well as achemically-modified bases or “universal bases,” which can be used tosubstitute for a standard pyrimidine within the nucleic acid moleculesof this disclosure.

The term “purine” as used herein refers to its meaning as is generallyaccepted in the art. The term generally refers to conventional purinebases, including the standard purine bases adenine and guanine. Inaddition, the term “purine” is contemplated to embrace non-standardpurine bases or acids, such as N₂-methylguanine, inosine, diaminopurineand the like, as well as chemically-modified bases or “universal bases,”which can be used to substitute for standard purines herein.

As described herein, a “base pair” can be formed between twonucleotides, a nucleotide and a modified nucleotide, two modifiednucleotides, a nucleotide and a nucleotide analog, two nucleotideanalogs, a nucleotide and a non-nucleotide substitute moiety, or twonon-nucleotide substitute moieties. In a specific embodiment, anon-nucleotide substitute can comprise any chemical moiety that iscapable of associating with a component of the cellular RNAi machinery,such as, for example, the PAZ domain, the PIWI domain, and/or otherArgonaute protein domains associated with the RISC. Non-traditionalWatson-Crick base pairs are also understood as “non-canonical basepairs,” which is meant any non-Watson Crick base pair, such asmismatches and/or wobble base pairs, including flipped mismatches,single hydrogen bond mismatches, trans-type mismatches, triple baseinteractions, and quadruple base interactions. Non-limiting examples ofsuch non-canonical base pairs include, but are not limited to, ACreverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AA N7amino, CC 2-carbonyl-amino(H1)-N3-amino(H2), GA sheared, UC4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AUreverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AAN1-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl,GA+carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-aminosymmetric, CC carbonyl-amino symmetric, CC N3-amino symmetric, UU2-carbonyl-imino symmetric, UU 4-carbonyl-imino symmetric, AA amino-N3,AA N1-amino, AC amino 2-carbonyl, AC N3-amino, AC N7-amino, AUamino-4-carbonyl, AU N1-imino, AU N3-imino, AU N7-imino, CCcarbonyl-amino, GA amino-N1, GA amino-N7, GA carbonyl-amino, GAN3-amino, GC amino-N3, GC carbonyl-amino, GC N3-amino, GC N7-amino, GGamino-N7, GG carbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GUcarbonyl-imino, GU imino-2-carbonyl, GU N7-imino, psiU imino-2-carbonyl,UC 4-carbonyl-amino, UC imino-carbonyl, UU imino-4-carbonyl, AC C2-H—N3,GA carbonyl-C2-H, UU imino-4-carbonyl 2 carbonyl-C5-H, AC amino(A)N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonylamino-2-carbonyl, and GU imino amino-2-carbonyl base pairs.

As used herein, the term “complementary” (or “complementarity”) refersto its meaning as is generally accepted in the art. The term generallyrefers to the formation or existence of hydrogen bond(s) between onenucleic acid sequence and another nucleic acid sequence by eithertraditional Watson-Crick or other non-traditional types of bonds asdescribed herein. With reference to exemplary nucleic acid molecules ofthe invention, complementarity can be found between a single-strandedRNAi of the invention and an RNA target sequence. The binding freeenergy for a nucleic acid molecule with its complementary sequence issufficient to allow the relevant function of the nucleic acid toproceed, e.g., RNAi activity. Determination of binding free energies fornucleic acid molecules is well known in the art (see, e.g., Turner etal., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986,Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am.Chem. Soc. 109:3783-3785).

As used herein, the term “perfectly complementary” (or “perfectcomplementarity”) between a first nucleic acid molecule (e.g., asingle-stranded RNAi molecule of the present invention) and the secondnucleic acid molecule (e.g., a target RNA sequence) means that all thecontiguous residues of the first nucleic acid sequence will hydrogenbond with the same number of contiguous residues in the second nucleicacid sequence. For example, two or more perfectly complementary nucleicacid strands can have the same number of nucleotides (i.e., have thesame length and form one double-stranded region with or without anoverhang), or have a different number of nucleotides (e.g., one strandmay be shorter but fully contained within a second strand). As anexample, if a single-stranded RNAi molecule of the invention has a firstnucleotide portion of only 1 nucleotide and a second nucleotide portionof 10 contiguous nucleotides, wherein all of the 10 nucleotides in thesecond nucleotide portion of the molecule base pair with the RNA targetsequence, the RNAi molecule is perfectly complementary with the RNAtarget sequence. The single nucleotide included in the first nucleotideportion is not included when determining the degree of complementaritybecause it is not within a contiguous chain of nucleotides. However, inthis example, if the first nucleotide portion contains 2 nucleotides,the RNAi molecule is perfectly complementary to the RNA target sequenceif the 2 nucleotides of the first nucleotide portion and the 10nucleotides of the second nucleotide portion base pair with the RNAtarget sequence.

Complementary nucleic acid molecules may have wrongly paired bases—thatis, bases that cannot form a traditional Watson-Crick base pair (i.e.,forming a hydrogen bond) or other non-traditional types of base pair(i.e., “mismatched” bases, formed or held together by non-traditionalforces that are not hydrogen bonds). The term “partially complementary”(or “partial complementarity”) between a first nucleic acid molecule(e.g., a single-stranded RNAi molecule of the present invention) andsecond nucleic acid molecule (e.g., a target RNA sequence) indicatesthat various mismatches or non-based paired nucleotides (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or more mismatches or non-based paired nucleotides)occur between the nucleotide sequences, which can result in, forexample, in bulges or loops. Such partial complementarity can berepresented by a percent (%) complementarity that is determined by thenumber of base paired nucleotides in relation to the total number ofnucleotides involved, e.g., about 50%, 60%, 70%, 80%, 90% etc. Forexample, a first nucleic acid molecule may have 10 nucleotides and asecond nucleic acid molecule may have 10 nucleotides, then base pairingof 5, 6, 7, 8, 9, or 10 nucleotides between the first and second nucleicacid molecules, which may or may not form a contiguous double-strandedregion, represents 50%, 60%, 70%, 80%, 90%, or 100% complementarity,respectively. In relation to the present invention, such partialcomplementarity is permitted to the extent that a single-stranded RNAimolecule of the invention maintains its function, for example theability to mediate sequence specific RNAi.

A first nucleic acid molecule can be “substantially complementary” to asecond nucleic acid. By “substantially complementary” it is meant that afirst nucleic acid sequence (e.g., a single-stranded RNAi molecule ofthe present invention) is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% complementary to a second nucleicacid sequence (e.g., a RNA target sequence). As used herein, a firstnucleic acid molecule can be both “partially complementary” and“substantially complementary” to a second nucleic acid molecule.

As used herein, the term “homologous” (or “homology”) refers to itsmeaning as is generally accepted in the art. The term generally refersto the number of nucleotides of the subject nucleic acid sequence thathas been matched to identical nucleotides of a reference nucleic acidsequence, typically as determined by a sequence analysis program (e.g.,Karlin and Altschul, 1990, PNAS 87:2264-2268; Karlin and Altschul, 1993,PNAS 90:5873-5877) or by visual inspection. The term “perfect homology”(or “perfectly homologous”) as used herein refers to complete (100%)homology or “identity” between a reference sequence and a subjectnucleic acid sequence. As used herein, the term “substantiallyhomologous” (or “substantial homology”) is meant that the subjectsequence shares at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) homologous nucleotides withthe nucleotides of the same nucleotide positions in a referencesequence.

The phrase “chemical modification” as used herein refers to its meaningas is generally accepted in the art. With reference to exemplary nucleicacid molecules of the invention, the term refers to any modification ofthe chemical structure of the nucleotides that differs from nucleotidesof native RNA. The term “chemical modification” encompasses theaddition, substitution, or modification of native RNA at the sugar,base, or internucleotide linkage, as described herein or as is otherwiseknown in the art. In certain embodiments, the term “chemicalmodification” can refer to certain forms of RNA that arenaturally-occurring in certain biological systems, for example2′-O-methyl modifications or inosine modifications.

The phrase “modified nucleotide” as used herein refers to its meaning asis generally accepted in the art. The term generally refers a nucleotidethat contains a modification in the chemical structure of the base,sugar and/or phosphate of the unmodified (or natural) nucleotide, as isgenerally known in the art. Non-limiting examples of modifiednucleotides are described herein and in U.S. application Ser. No.12/064,014.

“Percent modification” refers to its meaning as is generally accepted inthe art. As used herein, the term generally refers to the number ofnucleotides in a single-stranded RNA molecule of the invention that havebeen modified. The extent of chemical modifications will depend uponvarious factors well known to one skilled in the art (e.g., target RNA,off-target silencing, degree of endonuclease degradation).

The term “phosphorothioate” refers to its meaning as is generallyaccepted in the art. The term generally refers to an internucleotidephosphate linkage comprising one or more sulfur atoms in place of anoxygen atom. Hence, the term phosphorothioate refers to bothphosphorothioate and phosphorodithioate internucleotide linkages.

As used herein, the term “locked nucleic acid” (LNA) has the structureof the general Formula I:

X and Y are independently selected from the group consisting of —O—,—S—, —N(H)—, —N(R)—, —CH₂—, or —CH— (if part of a double bond), —CH₂—O—,CH₂—S—, CH₂—N(H)—, —CH₂—N(R)—, —CH₂—CH₂—, and CH₂—CH— (if part of adouble bond), —CH═CH—, where R is selected from hydrogen and C₁₋₄-alkyl;Z and Z* are independently selected from an internucleotide linkage, aterminal group or a protecting group; B constitutes a natural ornon-natural nucleobase; and the asymmetric groups may be found in eitherorientation.

The four chiral centers of Formula I, as shown, are in a fixedconfiguration, but their configurations are not necessary fixed. Assuch, the chiral centers can be found in different configurations, suchas those represented in Formula II (below). Thus, each chiral center inFormula 1 can exist in either R or S configuration. The definition of R(rectus) and S (sininster) are described in the IUPAC 1974Recommendations, Section E, Fundamental Stereochemistry: The rules canbe found in Pure Appl. Chem. 45, 13-30 (1976) and In “Nomenclature ofOrganic Chemistry” Pergamon, New York, 1979.

The terminal groups are selected independently among from hydrogen,azido, halogen, cyano, nitro, hydroxy, Prot-O—, Act-O—, mercapto,Prot-S—, Act-S—, C₁₋₆-alkylthio, amino, Prot-N(R^(H))—, Act-N(R^(H))—,mono- or di(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy,optionally substituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl,optionally substituted C₂₋₆-alkenyloxy, optionally substitutedC₂₋₆-alkynyl, optionally substituted C₂₋₆-alkynyloxy, monophosphate,monothiophosphate, diphosphate, dithiophosphate triphosphate,trithiophosphate, DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups,ligands, carboxy, sulphono, hydroxymethyl, Prot-O—CH₂—, Act-O—CH₂—,aminomethyl, Prot-N(R^(H))—CH₂—, Act-N(R^(H))—CH₂—, carboxy methyl,sulphonomethyl, where Prot is a protection group for —OH, —SH, and—NH(R^(H)), respectively, Act is an activation group for —OH, —SH, and—NH(R^(H)), respectively, and R^(H) is selected from hydrogen andC₁₋₆-alkyl.

The protection groups of hydroxy substituents comprises substitutedtrityl, such as 4,4′-dimethoxytrityloxy (DMT), 4-monomethoxytrityloxy(MMT), and trityloxy, optionally substituted 9-(9-phenyl)xanthenyloxy(pixyl), optionally substituted methoxytetrahydro-pyranyloxy (mthp),silyloxy such as trimethylsilyloxy (TMS), triisopropylsilyloxy (TIPS)₇tert-butyldimethylsilyloxy (TBDMS), triethylsilyloxy, andphenyldimethylsilyloxy, tert-butylethers, acetals (including two hydroxygroups), acyloxy such as acetyl or halogen substituted acetyls.

“Act” designates an activation group for —OH, —SH, and —NH(R^(H)),respectively. Such activation groups are, for example, selected fromoptionally substituted O-phosphoramidite, optionally substitutedO-phosphortriester, optionally substituted O-phosphordiester, optionallysubstituted H-phosphonate, and optionally substituted O-phosphonate.

B constitutes a natural or non-natural nucleobase and selected amongadenine, cytosine, 5-methylcytosine, isocytosine, pseudoisocytosine,guanine, thymine, uracil, 5-bromouracil, 5-propynyluracil,5-propyny-6-fluoroluracil, 5-methylthiazoleuracil, 6-aminopurine,2-aminopurine, inosine, diaminopurine, 7-propyne-7-deazaadenine,7-propyne-7-deazaguanine, and 2-chloro-6-aminopurine.

Preferably, the locked nucleic acid (LNA) used in a single-stranded RNAimolecule of the invention comprises a LNA structure according to any ofthe Formulas II:

wherein Y is —O—, —S—, —NH—, or N(R^(H)); Z and Z* are independentlyselected among an internucleotide linkage, a terminal group or aprotecting group; and B constitutes a natural or non-natural nucleobase.These exemplary LNA monomers and others, as well as their preparationare described in WO 99/14226 and subsequent applications, WO 00/56746,WO 00/56748, WO 00/66604, WO 00/125248, WO 02/28875, WO 2002/094250 andWO 2003/006475; the disclosure of all of which are incorporated hereinby reference.

The term “universal base” refers to its meaning as is generally acceptedin the art. The term generally refers to nucleotide base analogs thatform base pairs with each of the standard DNA/RNA bases with littlediscrimination among them, and is recognized by intracellular enzymes.See, e.g., Loakes et al., 1997, J. Mol. Bio. 270:426-435. Non-limitingexamples of universal bases include C-phenyl, C-naphthyl and otheraromatic derivatives, inosine, azole carbozamides, and nitroazolederivatives such as 3′-nitropyrrole, 4-nitroindole, 5-nitroindole, and6-nitroindole as known in the art. See, e.g., Loakes, 2001, NucleicAcids Res. 29:2437.

As used herein, the phrase “RNA interference” (also called “RNAi”herein) refers to its meaning as is generally accepted in the art. Theterm generally refers to the biological process of inhibiting,decreasing, or down-regulating gene expression in a cell, and which ismediated by short interfering nucleic acid molecules (e.g., siRNAs,miRNAs, shRNAs), see for example Zamore and Haley, 2005, Science309:1519-1524; Vaughn and Martienssen, 2005, Science 309:1525-1526;Zamore et al., 2000, Cell 101:25-33; Bass, 2001, Nature 411:428-429;Elbashir et al., 2001, Nature 411:494-498; and Kreutzer et al.,International PCT Publication No. WO 00/44895; Zernicka-Goetz et al.,International PCT Publication No. WO 01/36646; Fire, International PCTPublication No. WO 99/32619; Plaetinck et al., International PCTPublication No. WO 00/01846; Mello and Fire, International PCTPublication No. WO 01/29058; Deschamps-Depaillette, International PCTPublication No. WO 99/07409; and Li et al., International PCTPublication No. WO 00/44914; Allshire, 2002, Science 297:1818-1819;Volpe et al., 2002, Science 297:1833-1837; Jenuwein, 2002, Science297:2215-2218; and Hall et al., 2002, Science 297:2232-2237; Hutvagnerand Zamore, 2002, Science 297:2056-60; McManus et al., 2002, RNA8:842-850; Reinhart et al., 2002, Gene & Dev. 16:1616-1626; and Reinhart& Bartel, 2002, Science 297:1831). Additionally, the term “RNAinterference” (or “RNAi”) is meant to be equivalent to other terms usedto describe sequence-specific RNA interference, such aspost-transcriptional gene silencing, translational inhibition,transcriptional inhibition, or epigenetics. For example, single-strandedRNA molecules of the invention can be used to epigenetically silencegenes at either the post-transcriptional level or thepre-transcriptional level. In a non-limiting example, epigeneticmodulation of gene expression by single-stranded RNA molecules of theinvention can result from modification of chromatin structure ormethylation patterns to alter gene expression (see, for example, Verdelet al., 2004, Science 303:672-676; Pal-Bhadra et al., 2004, Science303:669-672; Allshire, 2002, Science 297:1818-1819; Volpe et al., 2002,Science 297:1833-1837; Jenuwein, 2002, Science 297:2215-2218; and Hallet al., 2002, Science 297:2232-2237). In another non-limiting example,modulation of gene expression by single-stranded RNA molecules of theinvention can result from cleavage of RNA (either coding or non-codingRNA) via RISC, or via translational inhibition, as is known in the artor modulation can result from transcriptional inhibition (see forexample Janowski et al., 2005, Nature Chemical Biology 1:216-222).

The terms “inhibit,” “down-regulate,” “reduce” or “knockdown” as usedherein refer to their meanings as are generally accepted in the art.With reference to exemplary single-stranded RNAi molecules of theinvention, the terms generally refer to the reduction in the (i)expression of a gene or target sequence and/or the level of RNAmolecules encoding one or more proteins or protein subunits, and/or (ii)the activity of one or more proteins or protein subunits, below thatobserved in the absence of the single-stranded RNAi molecules of theinvention. Down-regulation can also be associated withpost-transcriptional silencing, such as RNAi-mediated cleavage, or byalteration in DNA methylation patterns or DNA chromatin structure.Inhibition, down-regulation, reduction or knockdown with an RNAi agentcan be in reference to an inactive molecule, an attenuated molecule, anRNAi agent with a scrambled sequence, or an RNAi agent with mismatches.The phrase “gene silencing” refers to a partial or completeloss-of-function through targeted inhibition of an endogenous targetgene in a cell. As such, the term is used interchangeably with RNAi,“knockdown,” “inhibition,” “down-regulation,” or “reduction” ofexpression of a target gene.

To determine the extent of inhibition, a test sample (e.g., a biologicalsample from an organism of interest expressing the target gene(s) ortarget sequence(s) or a sample of cells in culture expressing the targetgene/sequence) can be contacted with an RNAi molecule that silences,reduces, or inhibits expression of a target gene or sequence. Expressionof the target gene/sequence in the test sample is compared to expressionof the target gene/sequence in a control sample (e.g., a biologicalsample from an organism of interest expressing the target gene/sequenceor a sample of cells in culture expressing the target gene/sequence)that is not contacted with the RNAi molecule. Control samples (i.e.,samples expressing the target gene/sequence) are assigned a value of100%. Silencing, inhibition, or reduction of expression of a targetgene/sequence is achieved when the value of the test sample relative tothe control sample is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%,50%, 45%, 40%, 35%, 30%, 25%, 20%, or 10%. Suitable assays include,e.g., examination of protein or mRNA levels using techniques known tothose of skill in the art, such as dot blots, Northern blots, in situhybridization, ELISA, microarray hybridization, immunoprecipitation,enzyme function, as well as phenotypic assays known to those of skill inthe art.

The phrase “improved RNAi activity” generally refers to an increase inRNAi activity measured in vitro and/or in vivo, where the RNAi activityis a reflection of either or both the ability of the RNAi agent tomediate RNAi and the stability of the RNAi agent.

The term “modulate” as used herein refers to its meaning as is generallyaccepted in the art. The term generally refers to when the expression ofa gene, or level of one or more RNA molecules (coding or non-coding), oractivity of one or more RNA molecules or proteins or protein subunits,is up-regulated or down-regulated, such that expression, level, oractivity is greater than or less than that observed in the absence ofthe molecule that effects modulation. For example, the term “modulate”in some embodiments can refer to inhibition and in other embodiments canrefer to potentiation or up-regulation, e.g., of gene expression.

The term “RNAi agent” or “RNAi molecule” refers to any nucleic acidmolecule capable of inhibiting or down-regulating gene expression orviral replication by mediating RNA interference (“RNAi”) or genesilencing in a sequence-specific manner. The RNAi agent can be adouble-stranded nucleic acid molecule comprising self-complementarysense (passenger) and antisense (guide) strands, wherein the antisensestrand comprises a nucleotide sequence that is complementary to anucleotide sequence in a target nucleic acid molecule or a portionthereof, and the sense strand comprises a nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof.An RNAi agent can be a single-stranded polynucleotide. While not wishingto be bound by theory, an RNAi agent may act by one or more of a numberof mechanisms, including post-transcriptional cleavage of a target mRNA,or pre-transcriptional or pre-translational mechanisms.

The term “single-stranded RNAi” or “ssRNAi” agent or molecule is an RNAiagent that is a single-stranded, nucleic acid-derived molecule having anucleotide sequence that is partially, substantially, or perfectlycomplementary to a nucleotide sequence in a target nucleic acid moleculeor a portion thereof. A second nucleotide sequence with which thesingle-stranded RNAi agent forms base-pairs is not present. Asingle-stranded RNAi molecule can further comprise a terminal phosphategroup located at one or both of the terminal ends, such as a5′-phosphate or a 5′,3′-diphosphate. An ssRNAi molecule/agent caninclude a miRNA or a miRNA mimetic. A single-stranded RNAi agent of theinvention can be loaded into or otherwise associated with RISC andparticipate in gene silencing via an RNAi mechanism. A single-strandedRNAi molecule of the invention can comprise substitutions,chemically-modified nucleotides, and non-nucleotides. A single-strandedRNAi molecule of the invention can comprise one or more or allribonucleotides. Certain embodiments of the invention includesingle-stranded RNAi molecules that comprise substitutions ormodifications in the backbone, sugars, bases, or nucleosides.

The term, “miRNA” or “microRNA” is used herein in accordance with itsordinary meaning in the art and refers to small, non-protein coding RNAmolecules that are expressed in a diverse array of eukaryotes, includingmammals, and are involved in RNA-based gene regulation. Mature, fullyprocessed miRNAs are about 15 to about 30 nucleotides in length. Arepresentative set of known, endogenous miRNA species is described inthe publicly available miRBase sequence database, described inGriffith-Jones et al., Nucleic Acids Research, 2004, 32:D109-D111 andGriffith-Jones et al., Nucleic Acids Research, 2006, 34:D 140-D144, andaccessible on the World Wide Web at the Wellcome Trust Sanger Institutewebsite. The mature, fully-processed miRNAs that are publicly availableon the miRBase sequence database are each incorporated by referenceherein. A representative set of miRNAs is also included herein in Table1, infra. Each mature miRNA is partially complementary to one or moremessenger RNA (mRNA) molecules, which are the targets of the miRNA,thereby regulating the expression of genes associated with the targets.

The term “miRNA mimetic,” as used herein, refers to a single-strandedRNA molecule that is a mimetic of a naturally-occurring miRNA in a cell.A miRNA mimetic is typically designed based on a corresponding,endogenous miRNA. A miRNA mimetic is capable of modulating theexpression of a target mRNA that is also regulated by a corresponding,naturally-occurring miRNA. A single-stranded RNAi molecule of thepresent invention that is also a miRNA mimetic can be loaded into orotherwise associated with RISC and participates in gene silencing via anRNAi mechanism. A miRNA mimetic of the invention can comprisesubstitutions, chemically-modified nucleotides, and non-nucleotides. AmiRNA mimetic of the invention can comprise one or more or allribonucleotides. Certain embodiments of the invention include miRNAmimetics that comprise substitutions or modifications in the backbone,sugars, bases, or nucleosides. A naturally-occurring miRNA in a cell isreferred to herein as “the corresponding miRNA,” “the endogenous miRNA,”or the “naturally-occurring miRNA.” A single-stranded miRNA mimetic ofthe invention that is provided to a cell is also understood to targetone or more target mRNAs that are also targeted by a corresponding,naturally-occurring miRNA. It is contemplated that a miRNA mimetic ofthe present invention introduced to a cell is capable of functioning asa naturally-occurring miRNA under appropriate conditions.

As used herein, the term “seed region” (also referred to herein as a“seed sequence”) refers to its meaning as is generally accepted in theart. The term generally refers to at least 6 consecutive nucleotideswithin nucleotide positions 1 to 10 of the 5′-end of anaturally-occurring mature miRNA, such as one selected from those listedin the publicly available miRBase sequence database(http://www.mirbase.org/) as of the filing date of the presentapplication and/or one selected from those listed in Table 1. The seedsequence nucleotides of positions 1 to 8 are capitalized in thesequences of Table 1. In a naturally-occurring miRNA, the seed regiontypically determines the target mRNA sequence to which the miRNA maybind and provide gene regulation. As such, multiple naturally-occurringmiRNAs can share a seed region or share substantial homology in the seedregions, and these miRNAs are members of the same miRNA family.

The term “siRNA” (also “short interfering RNA” or “small interferingRNA”) is given its ordinary meaning accepted in the art, generallyreferring to a duplex (sense and antisense strands) of complementary RNAoligonucleotides which may or may not comprise 3′ overhangs of about 1to about 4 nucleotides and which mediate RNA interference.

The term “siRNA mimetic” or “single-stranded siRNA mimetic,” as usedherein, refers to a single-stranded RNAi molecule that is a mimetic ofthe guide or antisense strand of a corresponding siRNA (either single ordouble-stranded). A siRNA mimetic is capable of modulating theexpression of a target RNA that is also regulated by the correspondingsiRNA and, thus, can be loaded into or otherwise associated with RISCand participates in gene silencing via an RNAi mechanism. Asingle-stranded siRNA mimetic of the invention can comprisesubstitutions, chemically-modified nucleotides, and non-nucleotides. AsiRNA mimetic of the invention can comprise one or more or allribonucleotides. Certain embodiments of the invention include siRNAmimetics that comprise substitutions or modifications in the backbone,sugars, bases, or nucleosides.

The term “gene” as used herein, especially in the context of “targetgene” for an RNAi agent, refers to the meaning as is generally acceptedin the art. The term generally refers to a nucleic acid (e.g., DNA orRNA) sequence that comprises partial length or entire length codingsequences necessary for the production of a polypeptide. The target genecan also include the UTR (i.e., untranslated region) or non-codingregion of the nucleic acid sequence. A gene or target gene can alsoencode a functional RNA (fRNA) or non-coding RNA (ncRNA), such as smalltemporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA),short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomalRNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Suchnon-coding RNAs can serve as target nucleic acid molecules for RNAinterference in modulating the activity of fRNA or ncRNA involved infunctional or regulatory cellular processes. Aberrant fRNA or ncRNAactivity leading to disease can therefore be modulated by the RNAiagents of the invention. RNAi agents targeting fRNA and ncRNA can alsobe used to manipulate or alter the genotype or phenotype of a subject,organism or cell, by intervening in cellular processes such as geneticimprinting, transcription, translation, or nucleic acid processing(e.g., transamination, methylation etc.). A target gene can be a genederived from a cell, an endogenous gene, a transgene, or exogenous genessuch as genes of a pathogen, for example a virus, which is present inthe cell after infection thereof. A cell containing a target gene can bederived from or contained in any organism, for example a plant, animal,protozoan, virus, bacterium, or fungus. Non-limiting examples of plantsinclude monocots, dicots, or gymnosperms. Non-limiting examples ofanimals include vertebrates or invertebrates. Non-limiting examples offungi include molds or yeasts. For a review, see for example Snyder andGerstein, 2003, Science 300:258-260.

The phrases “target site,” “target sequence,” and “target region,” asused herein, refer to their meanings as generally accepted in the art.The terms generally refer to a sequence within a target nucleic acidmolecule (e.g., mRNA) that is “targeted,” e.g., for cleavage mediated byan RNAi molecule that contains a sequence within its guide/antisenseregion that is partially, substantially, or perfectly complementary tothat target sequence. A “target site” for a single-stranded RNAimolecule of the present invention refers to a nucleic acid sequence thatis partially, substantially, or perfectly complementary to thesingle-stranded RNAi agent. The target site may be within a coding or anon-coding (i.e., untranslated) region of a target RNA. The target sitemay be the target site for an endogenous miRNA for which thesingle-stranded RNAi molecule is a mimetic, in which case the “targetsite” can also be referred to as a “miRNA target site” or a“corresponding miRNA target site.”

The phrase “sense region” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to a nucleotidesequence of an RNAi molecule having complementarity to an antisenseregion of the RNAi molecule. In addition, the sense region of an RNAimolecule can comprise a nucleic acid sequence having homology orsequence identity with a target nucleic acid sequence. The sense regionof an RNAi molecule is also referred to as the sense strand or thepassenger strand.

The phrase “antisense region” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to a nucleotidesequence of an RNAi molecule having complementarity to a target nucleicacid sequence. In addition, the antisense region of an RNAi molecule canoptionally comprise a nucleic acid sequence having complementarity to asense region of the RNAi molecule. The antisense region of an RNAimolecule is also referred to as the antisense strand or the guidestrand.

As used herein, the term “spacer” refers to any chemical group(s)capable of linking either two nucleotides and/or non-nucleotidesubstitute moieties. As used in the present invention, the “spacer” canconnect two nucleotides and/or non-nucleotide substitute moieties bytraditional phosphodiester bonds or non-phosphodiester connectors. Thespacer is typically an organic entity that is covalently bound to eachnucleotide or non-nucleotide substitute and is other than theinternucleotide linkages that form the backbone (i.e., the nucleobaseswhich form complementary hybrids).

As used herein, the term “alkyl” is intended to include a saturatedaliphatic hydrocarbon group, both branched and straight-chain, having aspecified number of carbon atoms. The term “alkyl” also refers tonon-aromatic cycloalkyl groups. Preferably, an alkyl group has from 1 to20 carbons (i.e., C₁-C₂₀). For example, C₁-C₁₀, as in “C₁-C₁₀ alkyl” isdefined to include groups having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbonsin a linear, branched or cyclic arrangement (i.e., cycloalkyl). The term“cycloalkyl” means a monocyclic saturated aliphatic hydrocarbon grouphaving the specified number of carbon atoms. For example, “alkyl”specifically includes methyl, ethyl, n-propyl, i-propyl, n-butyl,t-butyl, i-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and so on,as well as cycloalkyls, including cyclopropyl, methyl-cyclopropyl,2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, and so on. Analkyl group may be substituted, if indicated.

As used herein, the term “alkenyl” refers to a non-aromatic hydrocarbonradical, straight or branched, containing 2 or more carbon atoms and atleast 1 carbon to carbon double bond. The term “alkenyl” also refers tonon-aromatic cycloalkenyl groups. Preferably, the alkenyl group has from1 to 20 carbons (i.e., C1-C20). Alkenyl groups include, for example,ethenyl, propenyl, butenyl and cyclohexenyl. An alkenyl group maycontain double bonds and may be substituted, if indicated.

As used herein, the term “alkynyl” refers to a non-aromatic hydrocarbonradical, straight, or branched, containing 2 or more carbon atoms and atleast 1 carbon to carbon triple bond. The term “alkynyl” also refers tonon-aromatic cycloalkynyl groups. Up to 3 carbon-carbon triple bonds maybe present. Preferably, the alkynyl group has from 1 to 20 carbons(i.e., C₁-C₂₀). Alkynyl groups include, for example, ethynyl, propynyl,butynyl and cyclooctynl. An alkynyl group may contain triple bonds andmay be substituted, if indicated.

The term “aliphatic” as used herein in reference to a chemical grouprefers to an organic group composed of carbon and hydrogen which doesnot contain aromatic rings. Aliphatic structures can be cyclic and/orsaturated. The carbon atoms can be joined together in straight chains,branched chains, or non-aromatic rings. They can also be joined bysingle bonds (alkanes), double bonds (alkenes) or triple bonds(alkynes). Besides hydrogen, other elements can be bound to the carbonchain or substituted for a carbon within the chain, the most commonbeing oxygen, nitrogen, sulfur and chlorine.

The term “aromatic” as used herein in reference to a chemical grouprefers to an organic group containing a set of covalently-bound atomswith the following specific characteristics: (1) a delocalizedconjugated π system, most commonly an arrangement of alternating singleand double bonds; (2) coplanar structure, with all the contributingatoms in the same plane; (3) contributing atoms arranged in one or morerings; and, (4) a number of delocalized it electrons that is even, butnot a multiple of 4. An aromatic structure can be composed solely ofhydrocarbons (e.g., aryl). Other elements can be bound to or substitutedfor a carbon of the aromatic structure, the most common being oxygen,nitrogen, sulfur and chlorine (e.g., heteroaryl, substituted aryl,substituted heteroaryl).

The term “substituted” as used in reference to an aliphatic or aromaticorganic structure (e.g., an alkyl, alkenyl, alkynyl, aryl) refers to thepresence of additional chemical moieties and/or functional groups boundto the carbon chain. For example, a substituted hydrocarbon chain caninclude a hydrocarbon chain with a heteroatom (e.g., N, O, or S) boundto it. A substituted hydrocarbon chain can also include a hydrocarbonchain that is interrupted with a heteroatom. When substituted, thesubstituted group(s) is preferably, hydroxyl, halogen, cyano, C1-C4alkoxy, ═O, ═S, NO₂, SH, NH₂, or NR₁R₂, where R₁ and R₂ independentlyare H or C₁-C₄ alkyl. A substituted alkyl includes oligomers or polymersof ethylene oxide, including but not limited polyethylene glycol(“PEG”).

The term “non-nucleotide” or “non-nucleic acid” refers to any chemicalmolecule, moiety, group or compound that is not a nucleotide.

As used herein, the term “substitute non-nucleotide moiety” (or“non-nucleotide substitute moiety”) refers to a chemical moiety that iscapable of substituting for one or more nucleotides in a single-strandedRNAi molecule of the invention. Substitute non-nucleotide moieties aretypically those that allow for non-traditional base-pairing (i.e., notforming traditional hydrogen bonds). In certain embodiments, substitutenon-nucleotide moieties of the instant disclosure are those that arecapable of associating or otherwise interacting with one or morecomponents of the cellular RNAi machinery, including, for example, thePAZ domain, the PIWI domain and/or other Argonaute protein domainsassociated with the RISC.

The term “synthetic,” in certain embodiments herein, refers to nucleicacid molecules that are not produced naturally in a cell. Thesingle-stranded RNAi molecules of the invention are typically synthetic.

In certain embodiments, a single-stranded RNAi molecule of the inventionmay be isolated. The term “isolated,” as used herein in relation to anoligonucleotide, generally refers to a nucleic acid molecule that existsin a physical form differing from any nucleic acid molecules ofidentical sequence as found in nature. “Isolated” does not require,although it does not prohibit, that the nucleic acid be physicallyremoved from its native environment. For example, a nucleic acid can besaid to be “isolated” when it includes nucleotides and/orinternucleoside bonds not found in nature. A nucleic acid can be said tobe “isolated” when it exists at a purity not found in nature, wherepurity can be adjudged with respect to the presence of nucleic acids ofother sequences, with respect to the presence of proteins, with respectto the presence of lipids, or with respect to the presence of any othercomponent of a biological cell, or when the nucleic acid lacks sequencethat flanks an otherwise identical sequence in an organism's genome, orwhen the nucleic acid possesses sequence not identically present innature. A single-stranded RNAi molecule of the present invention can beisolated by virtue of its having been synthesized in vitro. It will beunderstood, however, that isolated nucleic acids may be subsequentlymixed or pooled together.

As used herein, “endogenous” refers its meaning as generally accepted inthe art. The term generally refers to any material from or producedinside an organism, cell, tissue or system. As used herein, an“endogenous miRNA” is a naturally-occurring miRNA in a cell, tissue,organism, including a mammal, such as, for example, a human. “Exogenous”generally refers to any material introduced from or produced outside anorganism, cell, tissue or system.

The term “expression” as used herein refers to its meaning as isgenerally accepted in the art. The term generally is defined as thetranscription and/or translation of a particular nucleotide sequencedriven by its promoter.

In some embodiments, it may be useful to know whether a cell expresses aparticular miRNA endogenously or whether such expression is affectedunder particular conditions or when it is in a particular disease state.Thus in some embodiments of the invention, methods include assaying acell or a sample containing a cell for the presence of one or moremarker genes or mRNA or other analyte indicative of the expression levelof a gene of interest. Consequently in some embodiments, methods includea step of generating an RNA profile for a sample. The term “RNA profile”or “gene expression profile” refers to a set of data regarding theexpression pattern for one or more gene or genetic marker in the sample(e.g., a plurality of nucleic acid probes that identify one or moremarkers).

By “capable of” is meant that, when RNAi activity is measured by asuitable in vivo or in vitro assay or method, a single-stranded RNAimolecule of the invention demonstrates at least 5% or more of theknockdown effect against a target sequence as compared to the knockdowneffect achieved by the corresponding single-stranded RNAi moleculewithout the internal, non-nucleotide spacer portion(s). Preferably, asingle-stranded RNAi molecule of the invention is capable of achieving25% or more, 35% or more, 50% or more, 55% or more, 60% or more, 65% ormore, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more,95% or more, 99% or more, or even 100% or more (i.e., equal or morepotent RNAi activity) knockdown of the target than a corresponding RNAimolecule against the same target (e.g., a naturally-occurring miRNA orpreviously-identified siRNA guide strand).

A “vector” is a replicon, such as a plasmid, phagemid, cosmid,baculovirus, bacmid, bacterial artificial chromosome (BAC), yeastartificial chromosome (YAC), as well as other bacterial, yeast, or viralvectors, to which another nucleic acid segment may be operativelyinserted so as to bring about replication or expression of the insertedsegment. “Expression vector” refers to a vector comprising expressioncontrol sequences operatively linked to a nucleotide sequence to beexpressed. An expression vector comprises sufficient cis-acting elementsfor expression; other elements for expression can be supplied by thehost cell or in an in vitro expression system. Expression vectorsinclude all those known in the art, such as cosmids, plasmids (e.g.,naked or contained in liposomes), and viruses (e.g., lentiviruses,retroviruses, adenoviruses, and adeno-associated viruses).

The terms “composition” or “formulation” as used herein refer to theirgenerally accepted meaning in the art. These terms generally refer to acomposition or formulation, such as in a pharmaceutically acceptablecarrier or diluent, in a form suitable for administration, e.g.,systemic or local administration, into a cell or subject, including, forexample, a human. Suitable forms, in part, depend upon the use or theroute of entry, for example oral, transdermal, inhalation, or byinjection. Such forms should not prevent the composition or formulationfrom reaching a target cell (i.e., a cell to which the negativelycharged nucleic acid is desirable for delivery). For example,compositions injected into the blood stream should be soluble. Otherfactors are known in the art, and include considerations such astoxicity and forms that prevent the composition or formulation fromexerting its effect. As used herein, pharmaceutical formulations includeformulations for human and veterinary use. Non-limiting examples ofagents suitable for formulation with the nucleic acid molecules of theinstant invention include: Lipid Nanoparticles (see for example Sempleet al., 2010, Nat Biotechnol. 28(2):172-6.); P-glycoprotein inhibitors(such as Pluronic P85); biodegradable polymers, such as poly(DL-lactide-coglycolide) microspheres for sustained release delivery(Emerich, D F et al, 1999, Cell Transplant 8:47-58); and loadednanoparticles, such as those made of polybutylcyanoacrylate. Othernon-limiting examples of delivery strategies for the nucleic acidmolecules of the instant invention include material described in Boadoet al., 1998, J. Pharm. Sci. 87:1308-1315; Tyler et al., 1999, FEBSLett. 421:280-284; Pardridge et al., 1995, PNAS USA. 92:5592-5596;Boado, 1995, Adv. Drug Delivery Rev., 15:73-107; Aldrian-Herrada et al.,1998, Nucleic Acids Res. 26:4910-4916; and, Tyler et al., 1999, PNAS96:7053-7058. A “pharmaceutically acceptable composition” or“pharmaceutically acceptable formulation” can refer to a composition orformulation that allows for the effective distribution of the nucleicacid molecules of the instant invention to the physical location mostsuitable for their desired activity.

The terms “patient,” “subject,” “individual” and the like are usedinterchangeably herein, and refer to any animal or cells or tissuesthereof, whether in vitro or in situ, amendable to the methods describedherein. They typically refer to an organism, which is a donor orrecipient of the single-stranded RNA molecules of this disclosure. Incertain non-limiting embodiments, the patient, subject or individual isa mammal or a mammalian cell. In other non-limiting embodiments, thepatient, subject or individual is a human or a human cell.

As used herein, the term “therapeutically effective amount” means anamount of a single-stranded RNAi molecule of the present disclosure thatis sufficient to result in a decrease in severity of disease symptoms,an increase in frequency or duration of disease symptom-free periods, ora prevention of impairment or disability due to the disease, in thesubject (e.g., a mammal or human) to which it is administered. One ofordinary skill in the art can determine such therapeutically effectiveamounts based on such factors such as the subject's size, the severityof symptoms, and the particular composition or route of administrationselected. For example, a therapeutically effective amount of asingle-strand RNAi molecule of the invention, individually, incombination, or in conjunction with other drugs, can be used oradministered at a therapeutically effective amount to a subject or byadministering to a particular cells under conditions suitable fortreatment, to, for example, decrease tumor size, or otherwise amelioratesymptoms associated with a particular disorder in the subject.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state. The term “treatment” as used hereinis meant to include therapeutic treatment as well as prophylactic, orsuppressive measures for diseases or disorders. Thus, for example, theterm “treatment” includes the administration of an agent prior to orfollowing the onset of a disease or disorder thereby preventing orremoving all signs of the disease or disorder. As another example,administration of the agent after clinical manifestation of the diseaseto combat the symptoms of the diseases is also comprised by the term“treatment.”

The term “parenteral” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers methods ortechniques of administering a molecule, drug, agent, or compound in amanner other than through the digestive tract, and includesepicutaneous, subcutaneous, intravascular (e.g., intravenous),intramuscular, or intrathecal injection or infusion techniques and thelike.

The phrase “systemic administration” as used herein refers to itsmeaning as is generally accepted in the art. The term generally refersin vivo systemic absorption or accumulation of drugs in the blood streamfollowed by distribution throughout the entire body.

Other objects, features and advantages of the present invention willbecome apparent from the detailed description. It should be understood,however, that the detailed description and the specific examples, whileindicating specific embodiments of the invention, are given by way ofillustration only, since various changes and modifications within thespirit and scope of the invention will become apparent to those skilledin the art from the detailed description.

B. Single-Stranded RNAi Molecules of the Invention

The instant disclosure provides single-stranded RNA molecules comprisingat least one internal, non-nucleotide spacer that links together twonucleotide portions of the molecule. Thus, a single-stranded RNAmolecule of the present invention is not a continuous stretch ofnucleotides but comprises more than one nucleotide portion separated byone or more non-nucleotide spacers, wherein the nucleotide portionscontain one or more nucleotides, non-nucleotide substitute moieties, ora combination thereof. A single-stranded RNA molecule of the inventionfunctions as a guide or antisense strand that is capable of inhibitinggene expression via an RNA interference mechanism and, thus, representsan RNAi agent. A single-stranded RNAi molecule of the inventioncomprises sequence that is partially, substantially or perfectlycomplementary to one or more RNA target sites in a cell.

A single-stranded RNAi molecule of the invention has a single-strandedoligonucleotide structure comprising (a) a nucleic acid portionseparated into two or more nucleotide portions, and (b) an internal (asopposed to “terminal”) spacer portion comprising at least onenon-nucleotide spacer portion, wherein the non-nucleotide spacerportion(s) covalently links two nucleotides, each within distinctnucleotide portions of the molecule. The nucleotide portions of asingle-stranded RNAi molecule of the invention are separated by thenon-nucleotide spacer portions, wherein each nucleotide portion containsat least one nucleotide.

In each embodiment of the invention, the nucleic acid portion of asingle-stranded RNAi molecule contains at least two nucleotide portions,a first nucleotide portion (N1) (e.g., a 5′-nucleotide portion) and asecond nucleotide portion (N2) (e.g., a 3′-nucleotide portion). Thenucleic acid portion of a single-stranded RNAi molecule of the inventioncan comprise more than two nucleotide portions (e.g., a third nucleotideportion (N3), a fourth nucleotide portion (N4) etc.). Within eachnucleotide portion of an RNAi molecule of the invention, the nucleotidesand/or non-nucleotide moieties are connected by phosphodiester bondsand/or non-phosphodiester connectors. Importantly, the nucleotideportions of a single-stranded RNAi molecule of the invention are notcomplementary to each other and, thus, said portions do not formsignificant base-pairing.

In each embodiment of the invention, the internal spacer portion of asingle-stranded RNAi molecule contains at least one non-nucleotidespacer portion (S1), referred to here in as a first non-nucleotidespacer portion. In one embodiment of the present invention, asingle-stranded RNAi molecule contains one internal, non-nucleotidespacer portion. The internal spacer portion of a single-stranded RNAimolecule of the invention can comprise more than a first non-nucleotidespacer portion (e.g., a second non-nucleotide spacer portion (S2), athird non-nucleotide spacer portion (S3) etc.). In another embodiment, asingle-stranded RNAi molecule contains two internal, non-nucleotidespacer portions.

The number of nucleotide portions within the nucleic acid portion of asingle-stranded RNAi molecule of the present invention is dependent onthe number of non-nucleotide spacer portions within the molecule, andvice versa. For example, if a single-stranded RNAi molecule contains twonon-nucleotide spacer portions, it will generally contain threenucleotide portions, as follows: 5′-(first nucleotide portion)-(firstnon-nucleotide spacer portion)-(second nucleotide portion)-(secondnon-nucleotide spacer portion)-(third nucleotide portion)-3′. Eachnon-nucleotide spacer portion of a single-stranded RNAi molecule of thepresent invention can contain one or more non-nucleotide spacers.

Single-stranded RNAi molecules of the invention have a single-strandedoligonucleotide structure and mediate RNA interference against a targetRNA. Single-stranded RNAi molecule of the invention can comprise: (a) anucleic acid portion comprising a first nucleotide portion (N1) and asecond nucleotide portion (N2), wherein said nucleic acid portioncomprises at least 8 nucleotides that can base pair with a target sitewithin a target RNA, and wherein the total number of nucleotides withinthe nucleic acid portion is from 8 to 26 nucleotides; and, (b) aninternal spacer portion comprising at least a first non-nucleotidespacer portion (S1) that covalently links the first and secondnucleotide portions. The first and second nucleotide portions are notself complementary. All of nucleotides (e.g., 8 to 26) of asingle-stranded RNAi molecule of the invention, all located within thenucleic acid portion, are distributed between the nucleotide portions ofthe molecule, wherein each nucleotide portion contains at least onenucleotide.

In one embodiment, a single-stranded RNAi molecule of the inventioncomprises a nucleic acid portion containing a total of from 8 to 26nucleotides or non-nucleotide substitute moieties (e.g., 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26nucleotides or non-nucleotide substitute moieties) distributed betweenthe nucleotide portions of the oligonucleotide, wherein at least 8(e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, or 26) of the nucleotides in the molecule can base pair with atarget site within a target RNA. For example, a single-stranded RNAimolecule of the invention may contain 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 total nucleotides, wherein 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26of those nucleotides base pair with a target RNA. In one embodiment, thenucleic acid portion of a single-stranded RNAi molecule of the inventioncontains a total of from 15 to 21 (e.g., 15, 16, 17, 18, 19, 20, or 21)nucleotides. In another embodiment, the nucleic acid portion of asingle-stranded RNAi molecule of the invention contains a total of from18 to 20 (e.g., 18, 19, or 20) nucleotides. In a further embodiment, thenucleic acid portion of a single-stranded RNAi molecule of the inventioncontains a total of 19 or 20 nucleotides.

The total number of nucleotides or non-nucleotide moieties, or acombination thereof, that make up the nucleotide portions of asingle-stranded RNAi molecule of the invention is distributed betweenthose portions of the molecule in any number of ways. As an example, asingle-stranded RNAi molecule comprising only one non-nucleotide spacerportion and two nucleotide portions (i.e., the first nucleotide portionand the second nucleotide portion) may have a total of 12 nucleotides.If the first nucleotide portion of the molecule contains a singlenucleotide (e.g., at the 5′-terminus of the molecule), the secondnucleotide portion of the molecule will contain 11 contiguousnucleotides. Alternatively, if the first nucleotide portion of themolecule contains 5 contiguous nucleotides, the second nucleotideportion of the molecule will contain 7 contiguous nucleotides. In eachexample, the total number of nucleotides in the molecule is 12. Thenucleotides within the nucleotide portions of a single-stranded RNAimolecule of the invention are not complementary to each other and, thus,said portions can not form substantial base-pairing. Within each of thenucleotide portions of the molecule, the nucleotides and/ornon-nucleotide moieties are connected by phosphodiester bonds and/ornon-phosphodiester connectors.

At least 8 nucleotides within the nucleic acid portion of asingle-stranded RNAi molecule of the invention can base pair with atarget sequence within a target RNA. Thus, the single-stranded RNAimolecules of the invention comprise a sequence of contiguous nucleotidesthat is partially, substantially or perfectly complementary to an RNAtarget site, including a naturally-occurring RNA target site. In oneembodiment, all of the contiguous nucleotides within the nucleic acidportion of a single-stranded RNAi molecule of the invention base pairwith a target sequence within a target RNA (i.e., perfectlycomplementary). In another embodiment, at least 50% of the contiguousnucleotides within the nucleic acid portion of a single-stranded RNAimolecule of the invention base pair with a target sequence within atarget RNA (i.e., substantially complementary). In another embodiment,8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26nucleotides within the nucleic acid portion of a single-stranded RNAimolecule of the invention base pair with a target sequence within atarget RNA.

In one embodiment, a single-stranded RNAi molecule of the invention hasa single-stranded oligonucleotide structure comprising: (a) twonucleotide portions, a first nucleotide portion (N1) and a secondnucleotide portion (N2); and, (b) one internal, non-nucleotide spacerportion (S1); wherein the oligonucleotide contains a total of from 8 to26 nucleotides or non-nucleotide substitute moieties (e.g., 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26nucleotides or non-nucleotide substitute moieties); and wherein at least8 of the nucleotides of the molecule can base pair with a target sitewithin a target RNA. The two nucleotide portions of a single-strandedRNAi molecule of this embodiment comprise, in sum, 8 to 26 (e.g., 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or, 26)nucleotides or non-nucleotide moieties, or a combination thereof, thatare distributed between the two nucleotide portions in any number ofways (as described above). In one embodiment, the non-nucleotide spacerportion contains one non-nucleotide spacer. In another embodiment, thenon-nucleotide spacer portion contains more than one non-nucleotidespacer (e.g., 2, 3, 4, or more). The spacer portion links the first andsecond nucleotide portions of the single-stranded RNAi molecule. Thus,the spacer portion is covalently linked to both the 3′-terminalnucleotide or non-nucleotide substitute moiety of the first nucleotideportion of the molecule and the 5′-terminal nucleotide or non-nucleotidesubstitute moiety of the second nucleotide portion of the molecule. Thespacer portion of the molecule can be covalently connected to thephosphate backbone of the nucleotide portions (i.e., through the freephosphate of the two, linked nucleotides) by either traditionalphosphodiester bonds or non-phosphodiester connectors.

In one embodiment, a single-stranded RNAi molecule of the inventioncomprises a contiguous nucleotide sequence that is partially,substantially or perfectly homologous to the guide strand of anaturally-occurring miRNA and, thus, functions as a miRNA mimetic. Inanother embodiment, a single-stranded RNAi molecule of the inventioncomprises a contiguous nucleotide sequence that is partially,substantially or perfectly homologous to either a single-stranded siRNAor the guide/antisense strand of a duplex siRNA and, thus, functions asa siRNA mimetic. The single-stranded siRNA or duplex siRNA may be knownto inhibit gene expression via an RNAi mechanism.

If a single-stranded RNAi molecule of the present invention is an analogof a naturally-occurring miRNA, the naturally-occurring miRNA isreferred to herein as “the corresponding miRNA,” and the single-strandedRNAi molecule represents a mimetic of the corresponding miRNA. Asingle-stranded miRNA mimetic of the present invention is designed basedon a corresponding, naturally-occurring miRNA, wherein at least onenon-nucleotide spacer portion is either inserted between two nucleotidesof the miRNA guide strand sequence or substituted for one or morenucleotides of the miRNA guide strand sequence. A single-stranded miRNAmimetic of the present invention can be an analog of a mature miRNAsequence publicly available in the miRBase database and/or includedwithin Table 1, infra (SEQ ID NOs: 1-1090).

In one embodiment, a single-stranded RNAi molecule as described hereinrepresents a miRNA mimetic, wherein the RNAi molecule comprises anucleic acid portion of two or more nucleotide portions and an internalspacer portion comprising at least one non-nucleotide spacer portion. Asdescribed above, if the nucleic acid portion of the molecule containsonly two nucleotide portions (i.e., a first nucleotide portion and asecond nucleotide portion), only one non-nucleotide spacer portion willbe present. If the nucleic acid portion of the molecule contains threenucleotide portions, two non-nucleotide spacer portions will be present.Each non-nucleotide spacer portion can comprise more than onenon-nucleotide spacer (e.g., 2, 3, 4 or more). In one embodiment, thenucleic acid portion of an miRNA mimetic of the invention consists offrom 8 to 26 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, or 26) nucleotides or non-nucleotide moieties, or acombination thereof, wherein at least 8 of the nucleotides can base pairwith a naturally-occurring miRNA target site. A contiguous nucleotidesequence within the nucleic acid portion of an miRNA mimetic of theinvention is partially, substantially or perfectly homologous to anaturally-occurring miRNA guide strand nucleotide sequence. In oneembodiment, a contiguous nucleotide sequence within a nucleic acidportion of a single-stranded RNAi molecule of the invention comprises 5to 8 (i.e., 5, 6, 7, or 8) contiguous nucleotides that are identical (orperfectly homologous) to the whole or a part of a seed sequence of anaturally-occurring miRNA. For example, in one embodiment, an 8consecutive nucleotide sequence within a nucleotide portion of asingle-stranded RNAi molecule is identical to all or a portion of theseed region of a naturally-occurring miRNA (see Table I, infra).

In one embodiment, a miRNA mimetic of the invention has a non-nucleotidespacer portion and two nucleotide portions, wherein the non-nucleotidespacer portion is inserted between two nucleotides of a corresponding,naturally-occurring miRNA sequence, separating the full-length,naturally-occurring miRNA into two distinct nucleotide portions. Inanother embodiment, more than one non-nucleotide spacer portion ispresent in a miRNA mimetic of the invention such that the nucleic acidportion of the miRNA mimetic is separated into more than two nucleotideportions. In such cases, the total nucleotide sequence of the miRNAmimetic is perfectly homologous to the corresponding,naturally-occurring miRNA nucleotide sequence. The difference betweenthe naturally-occurring miRNA and the miRNA mimetic in this embodimentis the presence of a non-nucleotide spacer portion.

In another embodiment, a miRNA mimetic of the invention comprises anon-nucleotide spacer portion that substitutes for one or morenucleotides of a naturally-occurring miRNA guide strand sequence. Forexample, one or more nucleotides may be first deleted from anaturally-occurring miRNA guide strand sequence, leaving a gap in thesequence and producing at least two distinct nucleotide portions. Anon-nucleotide spacer portion is then inserted into the gap, covalentlylinking the distinct nucleotide portions. Thus, in one embodiment, asingle-stranded RNAi molecule of the invention represents a miRNAmimetic wherein one or more internal, non-nucleotide spacer portionstakes the place of from one to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, or 12) nucleotides of a corresponding, naturally-occurring miRNAsequence (see SEQ ID NOs: 1-1090). A miRNA mimetic may contain more thanone non-nucleotide spacer portion. In one embodiment, a single-strandedmiRNA mimetic of the invention comprises at least one non-nucleotidespacer portion in the place of from one to 4 (e.g., 1, 2, 3, or 4)nucleotides of a naturally-occurring miRNA nucleotide sequence. Inanother embodiment, a single-stranded miRNA mimetic of the inventioncomprises at least one internal, non-nucleotide spacer portion in theplace of one or two nucleotides of a corresponding miRNA nucleotidesequence. The non-nucleotide spacer portion bridges the gap resultingfrom removal of the one or more nucleotides from a miRNA guide strandsequence, connecting by either traditional phosphodiester bonds ornon-phosphodiester connectors to the phosphate backbone of thenucleotide portions of the molecule.

Single-stranded RNAi molecules of the invention can also represent ananalog of the guide or antisense strand of a duplex or single-strandedsiRNA. The duplex or single-stranded siRNA may be known to inhibittarget gene expression, or have the potential of inhibiting target geneexpression, via an RNAi mechanism. In such a scenario, the siRNAcounterpart, and specifically the guide strand of the siRNA (whethersingle- or double-stranded), is referred to herein as “the correspondingsiRNA” or “the corresponding siRNA guide strand,” and thesingle-stranded RNAi molecule represents a mimetic of the correspondingsiRNA guide strand (i.e., “a single-stranded siRNA mimetic”). Asingle-stranded siRNA mimetic is designed based on the nucleotidesequence of a corresponding siRNA by either inserting one or moreinternal, non-nucleotide spacer portions within the nucleotide sequenceof the corresponding siRNA nucleotide sequence or substituting one ormore nucleotides of the corresponding siRNA nucleotide sequence with oneor more non-nucleotide spacer portions.

In one embodiment, a single-stranded RNAi molecule of the inventionrepresents a siRNA mimetic, wherein the nucleic acid portion of thesingle-stranded RNAi molecule comprises two or more nucleotide portions,and the internal spacer portion comprises at least one non-nucleotidespacer portion. As described above, if the nucleic acid portion of theRNAi molecule contains only two nucleotide portions (i.e., a firstnucleotide portion and a second nucleotide portion), only onenon-nucleotide spacer portion will be present. If the nucleic acidportion of the RNAi molecule contains three nucleotide portions, twonon-nucleotide spacer portions will be present. A non-nucleotide spacerportion may comprise more than one non-nucleotide spacer (e.g., 2, 3, 4or more). In one embodiment, the nucleic acid portion of a siRNA mimeticconsists of from 8 to 26 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, or 26) nucleotides or non-nucleotidemoieties, or a combination thereof, wherein at least 8 of thenucleotides can base pair with the an RNA target site. The nucleic acidportion of a siRNA mimetic of the invention comprises a contiguousnucleotide sequence that is partially, substantially or perfectlyhomologous to a corresponding siRNA guide strand nucleotide sequence.

In one embodiment, a siRNA mimetic of the invention has a non-nucleotidespacer portion and two nucleotide portions, wherein the non-nucleotidespacer portion is inserted between two adjacent nucleotides of thecorresponding siRNA nucleotide sequence, separating the correspondingsiRNA nucleotide sequence into two distinct nucleotide portions. Inanother embodiment, a siRNA mimetic of the invention can have more thanone non-nucleotide spacer portion such that the corresponding siRNAnucleotide sequence is separated into more than two nucleotide portions.In such cases, the total nucleotide sequence of the siRNA mimetic isperfectly homologous to the corresponding siRNA nucleotide sequence. Thedifference between the corresponding siRNA and the siRNA mimetic in thisembodiment is the presence of a non-nucleotide spacer region(s).

In another embodiment, a siRNA mimetic of the invention comprises one ormore non-nucleotide spacer portions that substitutes for one or morenucleotides of a corresponding siRNA guide strand nucleotide sequence.For example, one or more nucleotides may be first deleted from acorresponding siRNA nucleotide sequence, leaving a gap in the sequenceand producing at least two distinct nucleotide portions. Anon-nucleotide spacer portion is then inserted into the gap to link thedistinct nucleotide portions. Thus, in one embodiment, a single-strandedRNAi molecule of the present invention represents a siRNA mimeticcomprising at least one internal, non-nucleotide spacer portion, whereinsaid non-nucleotide spacer portion takes the place of from one to 4(e.g., 1, 2, 3, or 4) nucleotides of a corresponding siRNA nucleotidesequence. The siRNA mimetic may contain more than one non-nucleotidespacer portion. In another embodiment, a single-stranded RNAi moleculeof the present invention represents a siRNA mimetic comprising at leastone internal, non-nucleotide spacer portion, wherein said non-nucleotidespacer portion takes the place of one or two nucleotides of acorresponding siRNA nucleotide sequence. The non-nucleotide spacerportion(s) bridges the gap resulting from removal of the one or morenucleotides from the siRNA guide strand sequence, connecting by eithertraditional phosphodiester bonds or non-phosphodiester connectors to thephosphate backbone of the nucleotide portions of the molecule.

In another embodiment, single-stranded RNAi molecules of the inventioncan be designed de novo for the purpose of knocking down expression of aparticular RNA target, including a naturally-occurring RNA target. Inthis scenario, a target gene is first selected. One of skill in the artthen identifies a portion of said gene (i.e., the target site),generally between about 8 and about 26 nucleotides in length, to targetwith a single-stranded RNAi molecule for gene silencing. In oneembodiment of the invention, a contiguous nucleotide sequence within thenucleic acid portion of a single-stranded RNAi molecule described hereinis partially, substantially or perfectly complementary to the identifiedtarget site sequence and partially, substantially or perfectlyhomologous to the complement of the corresponding target site sequence.The counterpart sequence of the single-stranded RNAi molecule in thisscenario (i.e., a nucleotide sequence that is the complement of thetarget site sequence) is referred to herein as “the complement of thecorresponding target site sequence.” The single-stranded RNAi moleculecomprises two or more nucleotide portions and at least one internal,non-nucleotide spacer portion, as described in one or more of theembodiments above.

A single-stranded RNAi molecule of the present invention is capable ofproducing an RNA interference result. In the case of a single-strandedmiRNA mimetic of the invention, the molecule is capable of modulatingthe expression of a target mRNA that is also regulated by acorresponding naturally-occurring miRNA.

The single-stranded RNAi molecules of the disclosure can furthercomprise a terminal phosphate group located at one or both of theterminal ends, such as a 5′-phosphate or a 5′,3′-diphosphate. In someembodiments, a single-stranded RNAi molecule of the invention cancomprise substitutions, chemically-modified nucleotides, andnon-nucleotides. In certain other embodiments, a single-stranded RNAimolecule of the invention can comprise one or more or allribonucleotides. Certain embodiments of the invention includesingle-stranded RNAi molecules that comprise substitutions ormodifications in the backbone, sugars, bases, or nucleosides.

The internal, non-nucleotide spacer portion(s) of the single-strandedRNAi molecules of the disclosure, especially in situations where thetotal number of nucleotides in the resulting RNAi molecule is reducedcompared to a corresponding RNAi agent of which the single-stranded RNAimolecule is an analog (e.g., a naturally-occurring miRNA; the guidestrand of a siRNA with gene knockdown capability), reduces thesusceptibility of the single-stranded RNAi molecule to endonucleases.The internal, non-nucleotide spacer portion(s) can also limit the damageof exonucleases, ultimately helping to preserve the integrity of thesingle-stranded RNAi agent. The spacer portion also represents an easilyaccessible region for connecting one or more moieties of interest to theRNAi molecule (e.g., a chemical moiety that facilitates cellulardelivery). Therefore, even if the activity of a single-stranded RNAimolecule of this disclosure is somewhat reduced (e.g., by less thanabout 20%, or 30%, or even 40%) as compared to a correspondingsingle-stranded RNAi molecule without the spacer portion (e.g., anaturally-occurring miRNA; the guide strand of a previously identifiedsiRNA with gene knockdown capability), the overall activity of theanalog can be greater than that of its counterpart due to improvedstability or delivery of the molecule. Additionally, since the yield ofsynthesis is usually higher for shorter RNA strands, the cost oflarge-scale synthesis in connection with therapeutic applications mayalso be substantially reduced using the single-stranded RNAi moleculesof the present invention.

In one embodiment, a single-stranded RNAi molecule of the invention canbe represented or depicted by Formula III:

5′ N1-S1-N2 3′

wherein N1, representing a first nucleotide portion, consists of eitherone nucleotide or a contiguous stretch of nucleotides; S1, representinga non-nucleotide spacer portion, consists of one or more non-nucleotidespacers; and N2, representing a second nucleotide portion, consists ofeither one nucleotide or a contiguous stretch of nucleotides. The totalnumber of nucleotides in N1 and N2 is from 8 to 26 (e.g., 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26)nucleotides, and at least 8 nucleotides of the molecule can base pairwith a target site within a target RNA. The “nucleotide(s)” within N1and N2 are either nucleotides, modified nucleotides, nucleotide analogs,or non-nucleotides substitute moieties, or a combination thereof. In oneembodiment, individually, N1 and N2 can consist of between one and 25nucleotides, wherein the sum of N1 and N2 is from 8 to 26 (e.g., 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26)nucleotides. N1 and N2 are not self complementary and, thus, cannotparticipate in substantial base-pairing with each other. Within acontiguous stretches of nucleotides of the molecule, the nucleotides areconnected by phosphodiester bonds and/or non-phosphodiester connectors.The spacer portion (S1) is covalently attached to the 3′-terminalnucleotide of the first nucleotide portion (N1) and the 5′-terminalnucleotide of the second nucleotide portion of the molecule (N2). Forexample, the spacer portion can comprise one or more phosphoramiditespacers attached to the free phosphate group of the adjacent nucleotidesby a phosphodiester bond. The spacer portion of the molecule (S1) canconsist of a single non-nucleotide spacer or more than onenon-nucleotide spacers linked together. If there is more than onenon-nucleotide spacer within the S1 portion of the molecule, the spacerscan be either the same (i.e., having the same structure) or different(i.e., having different structures). In the case where twonon-nucleotide spacers are linked within the S1 portion of the molecule,each spacer is covalently attached to one nucleotide within the N1 andN2 portions of the molecule, respectively. If three non-nucleotidespacers are consecutively linked within the S1 portion of theoligonucleotide, the internal (second) spacer does not form a covalentbond with either the N1 or N2 portions of the molecule. Instead, theinternal spacer is covalently attached to the first and third spacers,linking them together.

In another embodiment, a single-stranded RNAi molecule of the inventioncan be represented or depicted by Formula IV:

5′ N1-S1-N2-S2-N3 3′

wherein N1, representing a first nucleotide portion, consists of eitherone nucleotide or a contiguous stretch of nucleotides; S1, representinga first non-nucleotide spacer portion, consists of one or morenon-nucleotide spacers; N2, representing a second nucleotide portion,consists of either one nucleotide or a contiguous stretch ofnucleotides; S2, representing a second non-nucleotide internal spacerportion, consists of one or more non-nucleotide spacers; and, N3,representing a third nucleotide portion, consists of either onenucleotide or a contiguous stretch of nucleotides. In one embodiment,the total number of nucleotides in N1, N2, and N3 is from 8 to about 26(e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, or 26) nucleotides, and at least 8 nucleotides of the molecule canbase pair with a target site within a target RNA. The “nucleotide(s)”within N1, N2 and N3 are either nucleotides, modified nucleotides,nucleotide analogs, or non-nucleotides substitute moieties, or acombination thereof. In one embodiment, individually, the nucleotideportions (N1, N2, N3) can consist of between one and 24 nucleotides,wherein the sum of nucleotides within the molecule is from 8 to 26nucleotides. The nucleotide portions of the RNAi molecule are not selfcomplementary and, thus, cannot participate in substantial base-pairingwith each other. Within each of the contiguous stretches of nucleotides,the nucleotides are connected by phosphodiester bonds and/ornon-phosphodiester connectors. The spacer portions are covalentlyattached to terminal nucleotides of the nucleotide portions of themolecule. In one embodiment, a spacer portion comprises one or morephosphoramidite spacers attached to the free phosphate groups ofadjacent nucleotides by phosphodiester bonds. Each spacer portion of themolecule can consist of a single non-nucleotide spacer or more than onenon-nucleotide spacer linked together. If there is more than onenon-nucleotide spacer within a spacer portion of the molecule, thespacers can be either the same (i.e., having the same structure) ordifferent (i.e., having different structures). When two non-nucleotidespacers are linked within a spacer portion of the molecule, each spaceris covalently attached to a terminal nucleotide within the adjacentnucleotide portions of the molecule. If three non-nucleotide spacers areconsecutively linked within a spacer portion of the molecule, theinternal (second) spacer does not form a covalent bond with a nucleotideportion of the molecule. Instead, the internal spacer is covalentlyattached to the first and third spacers, linking them together.

In one aspect of the invention, at least one nucleotide portion of asingle-stranded RNAi molecule described herein (e.g., N1, N2, or N3, asdescribed in Formulas III and/or IV) is a contiguous stretch ofnucleotides that consists of either from 1 to 20 (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) nucleotides,from 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20) nucleotides, from 10 to 20 (e.g., 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20) nucleotides, from 13 to 20 (e.g., 13, 14, 15, 16, 17,18, 19, or 20) nucleotides, from 5 to 15 (e.g., 5, 6, 7, 8, 9, 10, 11,12, 13, 14, or 15) nucleotides, or from 1 to 14 (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, or 14) nucleotides. In another aspect, thelength of at least one nucleotide portion of a single-stranded RNAimolecule of the invention is selected from the group consisting of 18contiguous nucleotides, 19 contiguous nucleotides, or 20 contiguousnucleotides. In a still further aspect, the length of at least onenucleotide portion of a single-stranded RNAi molecule of the inventionis selected from the group consisting of 13 contiguous nucleotides, 14contiguous nucleotides, or 15 contiguous nucleotides. The length of atleast one nucleotide portion of a single-stranded RNAi molecule of theinvention can be 18 nucleotides. The length of at least one nucleotideportion of a single-stranded RNAi molecule of the invention can be 19nucleotides. The length of at least one nucleotide portion of asingle-stranded RNAi molecule of the invention can be 20 nucleotides.The length of at least one nucleotide portion of a single-stranded RNAimolecule of the invention can be 21 nucleotides.

In one embodiment, a single-stranded RNAi molecule of the invention isrepresented by Formula III, wherein N1 consists of 18 contiguousnucleotides; S1 consists of a non-nucleotide spacer; and N2 consists oftwo contiguous nucleotides. In another embodiment, a single-strandedRNAi molecule of the invention is represented by Formula III, wherein N1consists of 19 contiguous nucleotides; S1 consists of a non-nucleotidespacer; and N2 consists of one nucleotide. In these embodiments, S1 canbe a C3- or C6-alkyl spacer.

In another aspect of the invention, a nucleotide portion of asingle-stranded RNAi molecule (e.g., N1, N2, or N3, as described byFormulas III and/or IV) is a contiguous stretch of nucleotides thatcomprises a sequence of at least 10 (e.g., 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 etc.) nucleotides that is substantially or perfectlycomplementary to an RNA target region. In another aspect, a nucleotideportion of a single-stranded RNAi molecule comprises a sequence of from5 to 8 contiguous nucleotides that is substantially or perfectlycomplementary to a RNA target region. In this portion of the invention,said nucleotide portion of the molecule is a contiguous stretch ofnucleotides that consists of either from 1 to 20 nucleotides, from 5 to20 nucleotides, from 10 to 20 nucleotides, from 13 to 20 nucleotides,from 5 to 15 nucleotides, or from 1 to 14 nucleotides. In anotheraspect, said nucleotide portion is 18, 19, or 20 contiguous nucleotidesin length. In a still further aspect, said nucleotide portion is 13, 14or 15 contiguous nucleotides in length. In another aspect, the length ofsaid nucleotide portion is selected from the group consisting of 18contiguous nucleotides, 19 contiguous nucleotides, or 20 contiguousnucleotides. In a still further aspect, the length of said nucleotideportion is selected from the group consisting of 13 contiguousnucleotides, 14 contiguous nucleotides, or 15 contiguous nucleotides.The length of said nucleotide portion can be 18 nucleotides. The lengthof said nucleotide portion can be 19 nucleotides. The length of saidnucleotide portion can be 20 nucleotides. The length of said nucleotideportion can be 21 nucleotides.

In one embodiment, a nucleotide portion of a single-stranded RNAimolecule of the invention comprises from 5 to 8 (e.g., 5, 6, 7, or 8)contiguous nucleotides that are identical (or perfectly homologous) tothe whole or a part of a seed sequence of a naturally-occurring miRNAsequence. In one embodiment, the naturally-occurring miRNA sequence is asequence recited in Table 1, infra. For example, in one embodiment, a6-nucleotide sequence within a nucleotide portion of a single-strandedRNAi molecule is identical to all or a portion of the seed region of anaturally-occurring miRNA sequence, including a naturally-occurringmiRNA sequence selected from Table 1.

In one embodiment, a single-stranded RNAi molecule of the invention canbe represented or depicted by Formula III or Formula IV. It should beappreciated that Formulas III and IV represent particular examples ofsingle-stranded RNAi molecules of the present invention. Additionalexamples encompassed by the present invention include, but are notlimited to, RNAi molecules having more than three nucleotide portions.

In one aspect of the present invention, a contiguous nucleotide sequencewithin the nucleic acid portion of a single-stranded RNAi molecule ispartially, substantially, or perfectly homologous to anaturally-occurring endogenous miRNA or to a guide strand of a siRNA. Inanother aspect of the invention, a contiguous nucleotide sequence withinthe nucleic acid portion of a single-stranded RNAi molecule ispartially, substantially, or perfectly complementary to a target sitewithin a RNA target sequence. In another embodiment, at least onenucleotide portion of a single-strand RNAi molecule of the disclosure ispartially, substantially or perfectly homologous to a region of anaturally-occurring endogenous miRNA or the guide strand of a siRNAand/or partially, substantially or perfectly complementary to a targetsite within a RNA target sequence.

The internal spacer portion of single-stranded RNAi molecules of theinvention comprises at least a first non-nucleotide spacer portion. Saidnon-nucleotide spacer portion comprises a chemical group, typically anorganic entity, covalently bound to, and thus linking, at least twonucleotides. The two nucleotides are within distinct nucleotide portionsof the molecule. There is no particular limitation in the length of anon-nucleotide spacer portion as long as it does not severely impact theability of the molecule to form traditional or non-traditionalWatson-Crick base pairing with an RNA target sequence and/or to mediateRNAi. A non-nucleotide spacer portion can connect two nucleotides and/ornon-nucleotide substitute moieties by traditional phosphodiester bondsor non-phosphodiester connectors. Single-stranded RNAi molecules of theinvention comprising non-phosphodiester based connectors linking thenucleotides and/or non-nucleotides to a spacer include, for example, apeptide-based connector, such as one linking the units of an oligopeptide nucleic acid (PNA) (see Boffa et al., 2000, Gene Ther. Mol.Biol. 5:47-53).

Various non-nucleotide moieties as are provided herein or otherwiseknown in the art can be included within the internal spacer portion ofthe single-stranded RNAi molecules of the invention. The non-nucleotidespacers comprised within the internal spacer portion of asingle-stranded RNAi molecule of the invention can include anynon-nucleic acid spacer capable of linking either two nucleotides and/ornon-nucleotide substitute moieties by either traditional phosphodiesterbonds or non-phosphodiester connectors. The spacer is typically analiphatic or aromatic organic entity and is other than theinternucleotide linkages that form the backbone of the oligonucleotide(i.e., the nucleobases which form complementary hybrids).

Non-limiting examples of non-nucleotide spacers include the following: apolyether, polyamine, polyamide, peptide, carbohydrate, lipid,polyhydrocarbon, or other polymeric compounds (e.g., polyethyleneglycols such as those having between 2 and 100 ethylene glycol units).Specific examples include those described by Seela and Kaiser, 1990,Nucleic Acids Res. 18:6353; Seela and Kaiser, 1987, Nucleic Acids Res.15:3113; Cload and Schepartz, 1991, J. Am. Chem. Soc. 113:6324;Richardson and Schepartz, 1991, J. Am. Chem. Soc. 113:5109; Ma et al.,1993, Nucleic Acids Res. 27:2585; Ma et al., 1993, Biochemistry 32:1751;Durand et al., 1990, Nucleic Acids Res. 18:6353; McCurdy et al., 1991,Nucleosides & Nucleotides 70:287; Jaschke et al., 1993, TetrahedronLett. 34:301; Ono et al., 1991, Biochemistry 30:9914; and others.

In one embodiment of the invention, a spacer is an alkyl, alkenyl oralkynyl chain of from one to 20 carbons (i.e., C1 to C20), preferablyfrom 1 to 12 carbons (i.e., C1 to C12), that is optionally substituted.The hydrocarbon chains can be substituted with additional chemicaland/or functional groups (e.g., a moiety that binds specifically to atarget molecule of interest).

A chemical moiety that provides additional functionality (e.g.,specifically binds to a target molecule of interest orfacilitates/enhances cellular delivery of the molecule) to asingle-stranded RNAi molecule may be a part of the spacer or covalentlyattached or linked thereto (e.g., substituted). For example, anadditional functional group can impart therapeutic activity to asingle-stranded RNAi molecule by assisting in transferring the RNAimolecule compounds across cellular membranes, altering thepharmacokinetics, and/or modulating the localization of RNAi moleculesof the invention.

Examples of specific conjugate molecules that may be incorporated withina non-nucleotide spacer itself and/or covalently attached thereto andare contemplated by the instant disclosure are small molecules, lipidsor lipophiles, terpenes, phospholipids, antibodies, toxins, cholesterol,a protein binding agent (e.g., a ligand for a cellular receptor that canfacilitate cellular uptake), a vitamin, negatively charged polymers andother polymers, for example proteins (e.g., human serum albumin),peptides, hormones, carbohydrates, polyethylene glycols, or polyamines,and those described in, for example, U.S. Patent Publication No.2005/0196781, and U.S. Patent Publication No. 2006/0293271, thedisclosures of which are incorporated herein by reference. Thesecompounds are expected to improve delivery and/or localization ofsingle-stranded RNAi molecules of the invention into a number of celltypes originating from different tissues, in the presence or absence ofserum (see Sullenger and Cech, U.S. Pat. No. 5,854,038). For example, aconjugate member can be naproxen, nitroindole (or another conjugate thatcontributes to stacking interactions), folate, ibuprofen, or a C5pyrimidine linker. In other embodiments, a conjugate member is aglyceride lipid conjugate (e.g., a dialkyl glyceride derivatives),vitamin E conjugates, or thio-cholesterols. In another embodiment, aconjugate molecule is a peptide that functions, when conjugated to asingle-stranded RNAi molecule, to facilitate delivery of the moleculeinto a target cell, or otherwise enhance delivery, stability, oractivity of the molecule when contacted with a biological sample.Exemplary peptide conjugate members for use within these aspects of thisdisclosure, include peptides PN27, PN28, PN29, PN58, PN61, PN73, PN158,PN159, PN173, PN182, PN202, PN204, PN250, PN361, PN365, PN404, PN453,and PN509 as described, for example, in U.S. Patent ApplicationPublication Nos. 2006/0040882 and 2006/0014289, and U.S. ProvisionalPatent Application No. 60/939,578, which are all incorporated herein byreference.

In one embodiment, a non-nucleotide spacer comprises a moiety thatspecifically binds to a target molecule. The target molecule can be anymolecule of interest. For example, the target molecule can be aligand-binding domain of a protein, thereby preventing or competing withthe interaction of the naturally-occurring ligand with the protein. Thisis a non-limiting example and those in the art will recognize that otherembodiments can be readily generated using techniques generally known inthe art (see, e.g., Gold et al, 1995, Annu. Rev. Biochem. 64:163; Brodyand Gold, 2000, J. Biotechnol. 74:5; Sun, 2000, Curr. Opin. Mol. Ther.2:100; Kusser, J., 2000, Biotechnol. 74:21; Hermann and Patel, 2000,Science 257:820; and Jayasena, 1999, Clinical Chem. 45:1628). The spacerportion of a single-stranded RNAi molecule of this disclosure can alsoconveniently be used to introduce functional chemical groups to an RNAimolecule to enhance properties associated with cellular delivery.

In one embodiment, a conjugate molecule or functional chemical moietyattached via a spacer region of a single-stranded RNAi molecule providesthe ability to administer said RNAi molecule to specific cell types,such as hepatocytes. For example, the asialoglycoprotein receptor(ASGPr) (Wu and Wu, 1987, J. Biol. Chem. 262:4429) is unique tohepatocytes and binds branched galactose-terminal glycoproteins, such asasialoorosomucoid (ASOR). Binding of such glycoproteins or syntheticglycoconjugates to the receptor takes place with an affinity thatstrongly depends on the degree of branching of the oligosaccharidechain, for example, triatennary structures are bound with greateraffinity than biatenarry or monoatennary chains (Baenziger and Fiete,1980, Cell 22: 611; Connolly et al., 1982, J. Biol. Chem. 257:939). Leeand Lee (1987, Glycoconjugate J. 4:317) obtained this high specificitythrough the use of N-acetyl-D-galactosamine as the carbohydrate moiety,which has higher affinity for the receptor compared to galactose. This“clustering effect” has also been described for the binding and uptakeof mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom etal., 1981, J. Med. Chem. 24: 1388). The use of galactose andgalactosamine based conjugates to transport exogenous compounds acrosscell membranes can provide a targeted delivery approach to the treatmentof liver disease. The use of bioconjugates can also provide a reductionin the required dose of therapeutic compounds required for treatment.Furthermore, therapeutic bioavailability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use ofbioconjugates of this disclosure

Conjugate molecules described herein can be attached to asingle-stranded RNAi molecule via non-nucleic acid linkers that arebiodegradable. The term “biodegradable linker,” as used in this context,refers to a non-nucleic acid linker molecule that is designed as abiodegradable linker to connect one molecule to another molecule, forexample, connecting a conjugate molecule to a single-stranded RNAimolecule of the invention. The biodegradable linker is designed suchthat its stability can be modulated for a particular purpose, such asdelivery to a particular tissue or cell type. The term “biodegradable,”as used herein, refers to degradation in a biological system, forexample enzymatic degradation or chemical degradation.

In one embodiment, a single-stranded RNAi molecule of the inventioncomprises an internal spacer portion comprising one or morenon-nucleotide spacer portions, wherein said one or more non-nucleotidespacer portions (e.g., S1 or S2 within Formula III and IV) comprise orconsist of a non-nucleotide spacer selected from the group consisting ofa C3, a C6, a C9, and a C12 aliphatic spacer. The number after the “C”indicates the number of carbon atoms in the core spacer structure (e.g.,if unsubstituted with additional chemical moieties). Said spacers can bealkyl, alkenyl, or alkynyl groups. Said spacers can also containphosphoramidite moieties to facilitate covalent linkage to the phosphatebackbone of the nucleotide portions of the molecule. In one embodiment,the spacer (S) portion is a C3 phosphoramidite spacer. In anotherembodiment, the spacer is a C6 phosphoramidite spacer. In a furtherembodiment, the C3, C6, C9, or C12 spacers are optionally substituted(e.g., with a targeting moiety).

One or more or all of the nucleotides within the nucleotide portions ofa single-stranded RNAi molecule of the disclosure may beribonucleotides, modified ribonucleotides, or suitable nucleotideanalogs. Incorporation of nucleotide analogs, such as various knownsugar, base, and backbone modifications, and LNA monomer units intodisrupted strands may significantly enhance serum stability and prolongtarget knockdown or expression regulatory effects. The single-strandedRNA molecules of the present invention can functionally accommodate andare compatible with various chemical modifications to varying degrees.For example, from 5% to 100% of the ribonucleotides of a single-strandedRNA molecule of the invention may be modified (e.g., 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100% of the ribonucleotides of the single-stranded RNAimolecules of the invention may be chemically modified or are replacedwith nucleotide analog residues). The improved properties conferred bythe functionally compatible chemical modifications to the sugar, baseand/or backbone, or by including suitable nucleotide analog residues,are of particular importance for application of these single-strandedRNAi molecules in vivo, for example, for use as a therapeutic agent oras a functional genomic tool.

In a further aspect, a single-stranded RNAi molecule of the invention,according to any of the embodiments herein, are capable of participatingin RNAi against a RNA target, including an endogenous RNA target. In oneembodiment, the endogenous RNA target is the target of anaturally-occurring miRNA. The inhibition of the RNA target may beachieved via the standard RNA-specific interference mechanism, includingmiRNA-dependent RNA interference. For example, the inhibition of a miRNAtarget may be by interaction (e.g., base-pairng, binding, etc.) with theuntranslated mRNA region, with which a corresponding endogenous miRNAinteracts, which effectuates the translational regulation of one or moredownstream genes. Alternatively, the inhibition of a miRNA target may beachieved via a siRNA-like interference mechanism wherein the binding ofthe miRNA target by the single-stranded RNAi molecule of the inventionthat is a miRNA mimetic results in the cleavage of the untranslatedmiRNA target. The single-stranded RNAi molecules of the invention mayalso inhibit mRNA target via a siRNA-like interference mechanism wherethe binding of the mRNA target in the sequence coding region (ratherthan in the non-coding untranslated region) by the single-stranded RNAimolecule of the invention results in cleavage of an mRNA target codingsequence.

C. Substituted and/or Modified Single-Stranded RNAi Molecules

The introduction of substituted and modified nucleotides intosingle-stranded RNAi molecules of the invention provides a tool forovercoming potential limitations of in vivo stability andbioavailability inherent to native RNA molecules (i.e., having standardnucleotides) that are exogenously delivered. In certain embodiments, theuse of substituted or modified single-stranded RNAi molecules of thisdisclosure can enable achievement of a given therapeutic effect at alower dose since these molecules may be designed to have an increasedhalf-life in a subject or biological samples (e.g., serum). Furthermore,certain substitutions or modifications can be used to improve thebioavailability of single-stranded RNAi molecules by targetingparticular cells or tissues or improving cellular uptake of thesingle-stranded RNAi molecules. Therefore, even if the activity of asingle-stranded RNAi molecule of this disclosure is somewhat reduced(e.g., by less than about 20%, or 30%, or even 40%) as compared to anunmodified or unsubstituted RNAi molecule of the same structure, theoverall activity of the substituted or modified RNAi molecule can begreater than that of its native counterpart due to improved stability ordelivery of the molecule. Substituted and/or modified single-strandedRNAi molecules can also minimize the possibility of activating aninterferon response in, for example, humans.

In certain embodiments, single-stranded RNAi molecules of the inventioncomprise ribonucleotides at about 5% to about 95% of the nucleotidepositions. For example, from one to all (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 26, or27) of the ribonucleotides of the single-stranded RNAi molecules of theinvention can be modified.

In related embodiments, a single-stranded RNAi molecule according to theinstant disclosure comprises one or more natural or syntheticnon-standard nucleoside. In related embodiments, the non-standardnucleoside is one or more deoxyuridine, L- or D-locked nucleic acid(LNA) molecule (e.g., a 5-methyluridine LNA) or substituted LNA (e.g.,having a pyrene), or a universal-binding nucleotide, or a G clamp, orany combination thereof. In certain embodiments, the universal-bindingnucleotide can be C-phenyl, C-naphthyl, inosine, azole carboxamide,1-β-D-ribofuranosyl-4-nitro indole, 1-β-D-ribofuranosyl-5-nitroindole,1-β-D-ribofuranosyl-6-nitroindole, or1-β-D-ribofuranosyl-3-nitropyrrole.

Substituted or modified nucleotides, which can be present in thesingle-stranded RNAi molecules of the invention, comprise modified orsubstituted nucleotides having characteristics similar to natural orstandard ribonucleotides. For example, this disclosure featuressingle-stranded RNAi molecules comprising nucleotides having a Northernconformation (see, e.g., Northern pseudorotation cycle, Saenger,Springer-Verlag ed., 1984), which are known to potentially impartresistant to nuclease degradation while maintaining the capacity tomediate RNAi, at least when applied to siRNA molecules. Exemplarynucleotides having a Northern configuration include locked nucleic acid(LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl)nucleotides), 2′-methoxyethyl (MOE) nucleotides, 2′-methyl-thio-ethyl,2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azidonucleotides, 5-methyluridines, or 2′-O-methyl nucleotides). In any ofthese embodiments, one or more substituted or modified nucleotides canbe a G clamp (e.g., a cytosine analog that forms an additional hydrogenbond to guanine, such as 9-(aminoethoxy)phenoxazine). See, e.g., Lin andMateucci, 1998, J. Am. Chem. Soc. 720:8531.

In certain embodiments, the 5′-terminal end of single-stranded RNAimolecules of the invention is phosphorylated. In any of the embodimentsof single-stranded RNAi molecules described herein, the molecule canfurther comprise a terminal phosphate group, such as a 5′-phosphate (seeMartinez et al., 2002, Cell 110:563; Schwarz et al., 2002, Mole. Cell70:537) or a 5′3′-diphosphate.

In another aspect, a single-stranded RNAi molecule of the inventioncomprises one or more 5′- and/or a 3′-cap structure at the terminal endsof the molecule. By “cap structure” is meant chemical modifications,which have been incorporated into the ends of oligonucleotide (see, forexample, Matulic-Adamic et al., U.S. Pat. No. 5,998,203, incorporated byreference herein). These terminal modifications can protect certainnucleic acid molecules from exonuclease degradation, and can impartcertain advantages in delivery and/or cellular localization. Innon-limiting examples: a suitable 5′-cap can be one selected from thegroup comprising inverted abasic residue (moiety); 4′,5′-methylenenucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide;carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide,3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety;3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety;1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexylphosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; orbridging or non-bridging methylphosphonate moiety.

In another non-limiting example, a suitable 3′-cap can be selected froma group comprising, 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclicnucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate;3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecylphosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide;L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties. For more details, seeBeaucage and Iyer, 1993, Tetrahedron 49:1925, which is incorporated byreference herein.

In certain embodiments, this disclosure features modifiedsingle-stranded RNAi molecules comprising phosphate backbonemodifications, including, for example, one or more phosphorothioate,phosphorodithioate, methylphosphonate, phosphotriester, morpholino,amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate,sulfonamide, sulfamate, formacetal, thioformacetal, or alkylsilyl,substitutions. For a review of oligonucleotide backbone modifications,see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis andProperties, in Modern Synthetic Methods, VCH, 331; Mesmaeker et al.,1994, ACS 24-39.

In further embodiments, a single-stranded RNAi molecule comprises one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, or 26) 2′-sugar substitutions, such as a2′-deoxy, 2′-O-2-methoxyethyl, 2′-O-methoxyethyl, 2′-O-methyl,2′-halogen (e.g., 2′-fluoro), 2′-O-allyl, or the like, or anycombination thereof. In still further embodiments, a single-strandedRNAi molecule comprises a terminal cap substituent at one or bothterminal ends, such as, for example, an alkyl, abasic, deoxy abasic,glyceryl, dinucleotide, acyclic nucleotide, inverted deoxynucleotidemoiety, or any combination thereof. In certain embodiments, at least one5′-terminal-end ribonucleotide has a 2′-sugar substitution.

In other embodiments, a single-stranded RNAi molecule comprises one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, or 26) substitutions in the sugarbackbone, including any combination of ribosyl, 2′-deoxyribosyl, atetrofuranosyl (e.g., L-α-threofuranosyl), a hexopyranosyl (e.g.,β-allopyranosyl, β-altropyranosyl and β-glucopyranosyl), apentopyranosyl (e.g., β-ribopyranosyl, α-lyxopyranosyl, β-xylopyranosyland α-arabinopyranosyl), a carbocyclic analog, a pyranose, a furanose, amorpholino, or analogs or derivatives thereof.

In yet other embodiments, a single-stranded RNAi molecule comprises atleast one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) modified internucleosidelinkage, such as independently a phosphorothioate, chiralphosphorothioate, phosphorodithioate, phosphotriester,aminoalkylphosphotriester, methyl phosphonate, alkyl phosphonate,3′-alkylene phosphonate, 5′-alkylene phosphonate, chiral phosphonate,phosphonoacetate, thiophosphonoacetate, phosphinate, phosphoramidate,3′-amino phosphoramidate, aminoalkylphosphoramidate,thionophosphoramidate, thionoalkylphosphonate,thionoalkylphosphotriester, selenophosphate, boranophosphate linkage, orany combination thereof.

A single-stranded RNAi molecule can comprise one or more modifiedinternucleotide linkages at the 3′-terminal end, the 5′-terminal end, orboth of the 3′-terminal and 5′-terminal ends of the molecule. In oneembodiment, a single-stranded RNAi molecule of the invention has onemodified internucleotide linkage at the 3′-terminal end, such as aphosphorothioate linkage. An exemplary single-stranded RNAi moleculecomprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioateinternucleotide linkages. A further exemplary single-stranded RNAimolecule comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreconsecutive phosphorothioate internucleotide linkages at, for example,the 5′-terminal end of the molecule. In yet another exemplarysingle-stranded RNAi molecule, there can be one or more pyrimidinephosphorothioate internucleotide linkages. In a further exemplarysingle-stranded RNAi molecule, there can be one or more purinephosphorothioate internucleotide linkages.

Many exemplary modified nucleotide bases or analogs thereof useful insingle-stranded RNAi molecules of the instant disclosure include5-methylcytosine; 5-hydroxymethylcytosine; xanthine; hypoxanthine;2-aminoadenine; 6-methyl, 2-propyl, or other alkyl derivatives ofadenine and guanine; 8-substituted adenines and guanines (e.g., 8-aza,8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, or the like);7-methyl, 7-deaza, and 3-deaza adenines and guanines; 2-thiouracil;2-thiothymine; 2-thiocytosine; 5-methyl, 5-propynyl, 5-halo (e.g.,5-bromo or 5-fluoro), 5-trifluoromethyl, or other 5-substituted uracilsand cytosines; and 6-azouracil. Further useful nucleotide bases can befound in Kurreck, 2003, Eur. J. Biochem. 270:1628; Herdewijn, 2000,Guide Nucleic Acid Develop. 10:297; Concise Encyclopedia of PolymerScience and Engineering, pp. 858-859, Kroschwitz, J. L, ed. John Wiley &Sons, 1990; U.S. Pat. No. 3,687,808, and similar references, all ofwhich are incorporated by reference herein.

Certain substituted or modified nucleotide base moieties are alsocontemplated. These include 5-substituted pyrimidines; 6-azapyrimidines;and N-2, N-6, or 0-6 substituted purines (e.g., 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine). Further, for example,5-methyluridine and 5-methylcytosine substitutions are known to increasenucleic acid duplex stability, which can be combined with 2′-sugarmodifications (e.g., 2′-O-methyl or 2′-methoxyethyl) or internucleosidelinkages (e.g., phosphorothioate) that provide the desired nucleaseresistance to the modified or substituted single-stranded RNAi molecule.

In further embodiments, at least one pyrimidine of a single-strandedRNAi molecule of the invention is a locked nucleic acid (LNA) in theform of a bicyclic sugar. In a related embodiment, the LNA comprises abase substitution, such as a 5-methyluridine LNA or2-thio-5-methyluridine LNA. In further embodiments, a ribose of thepyrimidine nucleoside or the internucleoside linkage can be optionallymodified.

In any of these embodiments, one or more substituted or modifiednucleotides can be a G clamp (e.g., a cytosine analog that forms anadditional hydrogen bond to guanine, such as 9-(aminoethoxy)phenoxazine). See, e.g., Lin and Mateucci, 1998, Nucleic Acids Res.19:3111.

In any of the embodiments described herein, a single-stranded RNAimolecule may include multiple types of modifications. For example, asingle-stranded RNAi molecule having at least one ribothymidine or2-thioribothymidine can further comprise at least one LNA, 2′-methoxy,2′-fluoro, 2′-deoxy, phosphorothioate linkage, an inverted base terminalcap, or any combination thereof. In certain exemplary embodiments, asingle-stranded RNAi molecule can comprise one or more or all uridinessubstituted with ribothymidine and have up to about 75% LNAsubstitutions. In other exemplary embodiments, a single-stranded RNAimolecule can comprise from one or more or all uridines substituted withribothymidine and have up to about 25% 2′-methoxy substitutions. Instill other exemplary embodiments, a single-stranded RNAi molecule cancomprise one or more or all uridines substituted with ribothymidine andhave up to about 100% 2′-fluoro substitutions.

Within certain aspects, the present disclosure also providessingle-stranded RNAi molecules comprising one or more universal basenucleotides. The term “universal base” as used herein refers tonucleotide base analogs that form base pairs or hydrogen bondednucleotide pairs with more than one types of nucleotides. Non-limitingexamples of universal bases include C-phenyl, C-naphthyl and otheraromatic derivatives, inosine, azole carboxyamides, and nitroazolederivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and6-nitroindole as known in the art (see, e.g., Loakes, 2001, NucleicAcids Research 29:2437-2447). In certain aspects, a single-stranded RNAimolecule disclosed herein can include about 1 to about 10 universal basenucleotides, so long as the resulting RNAi molecule remains capable ofmodulating one or more of its endogenous targets.

D. Synthesis of Single-Stranded RNAi Molecules

Exemplary molecules of the instant disclosure can be obtained using anumber of techniques known to those of skill in the art. For example,the RNAi molecules of the invention can be chemically synthesized,recombinantly produced (e.g., encoded by plasmid), or a combinationthereof.

Oligonucleotides or individual contiguous stretches of nucleotides(e.g., certain modified oligonucleotides or portions of oligonucleotideslacking ribonucleotides) are synthesized using protocols known in theart, for example, as described in Caruthers et al., 1992, Methods inEnzymol. 211:3; Thompson et al, PCT Publication No. WO 99/54459; Wincottet al., 1995, Nucleic Acids Res. 23:2677; Wincott et al., 1997, MethodsMol. Bio. 74:59; Brennan et al., 1998, Biotechnol. Bioeng. 67:33; andBrennan, U.S. Pat. No. 6,001,311. The synthesis of oligonucleotidesmakes use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end.Synthesis of RNA without modifications, including certainsingle-stranded RNAi molecules thereof of this disclosure, can be madeusing the procedure as described in Usman et al., 1987, J. Am. Chem.Soc. 109:7845; Scaringe et al., 1990, Nucleic Acids Res. 18:5433; andWincott et al., 1995, Nucleic Acids Res. 23:2677; and Wincott et al.,1997, Methods Mol. Bio. 74:59. In certain embodiments, the nucleotideportions of the single-stranded RNAi molecules of the present disclosurecan be synthesized separately and joined together with thenon-nucleotide spacer portions post-synthetically, for example, byligation (Moore et al., 1992, Science 256:9923; Draper et al., PCTPublication No. WO 93/23569; Shabarova et al., 1991, Nucleic Acids Res.19:4247; Bellon et al., 1997, Nucleosides & Nucleotides 16:951; Bellonet al., 1997, Bioconjugate Chem. 8:204). In a further embodiment, thenucleotide portion of a single-stranded RNAi molecule of this disclosurecan be made as single or multiple transcription products expressed by apolynucleotide (DNA or RNA) vector encoding one or more contiguousstretches of RNAs and directing their expression within host cells. Thenucleotide portions are then isolated and joined by ligation with anon-nucleotide spacer portion.

In some embodiments, pol III based constructs are used to expressnucleic acid molecules of the invention. Transcription of thesingle-stranded RNAi molecule sequences can be driven from a promoterfor eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), orRNA polymerase III (pol III). (see for example, Thompson, U.S. Pat. Nos.5,902,880 and 6,146,886). (See also, Izant and Weintraub, 1985, Science,229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83,399; Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5;Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Dropulic etal., 1992, J. Virol., 66, 1432-41; Weerasinghe et al., 1991, J. Virol.,65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89,10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver etal., 1990 Science, 247, 1222-1225; Thompson et al., 1995, Nucleic AcidsRes., 23, 2259; Good et al., 1997, Gene Therapy, 4, 45. Transcripts frompol II or pol III promoters are expressed at high levels in all cells;the levels of a given pol II promoter in a given cell type depends onthe nature of the gene regulatory sequences (enhancers, silencers, etc.)present nearby. Prokaryotic RNA polymerase promoters are also used,providing that the prokaryotic RNA polymerase enzyme is expressed in theappropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci.USA, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72;Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990,Mol. Cell. Biol., 10, 4529-37). Several investigators have demonstratedthat nucleic acid molecules expressed from such promoters can functionin mammalian cells (e.g. Kashani-Sabet et al., 1992, Antisense Res.Dev., 2, 3-15; Ojwang et al., 1992, Proc. Natl. Acad. Sci. US A, 89,10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Yu et al.,1993, Proc. Natl. Acad. Sci. US A, 90, 6340-4; L'Huillier et al., 1992,EMBO J., 11, 4411-8; Lisziewicz et al., 1993, Proc. Natl. Acad. Sci.U.S.A, 90, 8000-4; Thompson et al., 1995, Nucleic Acids Res., 23, 2259;Sullenger & Cech, 1993, Science, 262, 1566). More specifically,transcription units such as the ones derived from genes encoding U6small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA areuseful in generating high concentrations of desired RNA molecules(Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberget al., 1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat.No. 5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al.,International PCT Publication No. WO 96/18736. The above transcriptionunits can be incorporated into a variety of vectors for introductioninto mammalian cells, including but not restricted to, plasmid DNAvectors, viral DNA vectors (such as adenovirus or adeno-associated virusvectors), or viral RNA vectors (such as retroviral or alphavirusvectors) (for a review see Couture and Stinchcomb, 1996, supra).

Chemically synthesizing nucleic acid molecules with substitutions ormodifications (base, sugar, phosphate, or any combination thereof) canimpart resistance to degradation by serum ribonucleases, which may leadto increased potency and other pharmacological and therapeutic benefits.See, e.g., Eckstein et al., PCT Publication No. WO 92/07065; Perrault etal., 1990, Nature 344:565; Pieken et al., 1991, Science 253:314; Usmanand Cedergren, 1992, Trends in Biochem. Sci. 77:334; Usman et al., 1994,Nucleic Acids Symp. Ser. 31:163; Beigelman et al., 1995, J. Biol Chem.270:25702; Burlina et al., 1997, Bioorg. Med. Chem. 5:1999; Karpeisky etal., 1998, Tetrahedron Lett. 39:1131; Earnshaw and Gait, 1998,Biopolymers (Nucleic Acid Sciences) 48:39; Verma and Eckstein, 1998,Annu. Rev. Biochem. 67:99; Herdewijn, 2000, Guide Nucleic Acid Drug Dev.10:291; Kurreck, 2003, Eur. J. Biochem. 270:1628; Dorsett and Tuschl,2004, Nature Rev. Drug Discov. 3:318; Rossi et al., PCT Publication No.WO 91/03162; Usman et al., PCT Publication No. WO 93/15187; Beigelman etal., PCT Publication No. WO 97/26270; Woolf et al., PCT Publication No.WO 98/13526; Sproat, U.S. Pat. No. 5,334,711; Usman et al., U.S. Pat.No. 5,627,053; Beigelman et al., U.S. Pat. No. 5,716,824; Otvos et al.,U.S. Pat. No. 5,767,264; Gold et al., U.S. Pat. No. 6,300,074. Each ofthe above references discloses various substitutions and chemicalmodifications to the base, phosphate, or sugar moieties of nucleic acidmolecules, which can be used in the single-stranded RNAi moleculesdescribed herein.

E. Methods for Designing a Single-Stranded RNAi Molecule

As described herein, the single-stranded RNA molecules of the presentinvention are capable of inhibiting the expression of a target sequencevia an RNAi mechanism. In one embodiment, the single-stranded RNAimolecules can be designed based on a previously identified RNAi agentpossessing a desired knockdown function (e.g., siRNA, miRNA). Forexample, if a single-stranded RNAi molecule of the present invention isa miRNA mimetic, it is derived from a corresponding, naturally-occurringmiRNA molecule (see Table 1) or an analog thereof (e.g., a chemicallymodified form). As of the filing date of the present application, over3000 miRNA molecules endogenous to a variety of species can be found inpublically available databases (see, e.g., the publicly availablemiRBase sequence database as described in Griffith-Jones et al., 2004,Nucleic Acids Research 32:D109-D111 and Griffith-Jones et al., 2006,Nucleic Acids Research 34:D 140-D144, accessible on the World Wide Webat the Wellcome Trust Sanger Institute website). Table 1 herein containsa list of 1090 mature human miRNA sequences (SEQ ID NO: 1-1090). Inanother example, a single-stranded RNAi molecule of the presentinvention may be derived from a previously identified siRNA either knownto inhibit expression of a target sequence of choice or has thepotential of inhibiting expression of a target mRNA sequence.Specifically, a single-stranded RNAi molecule that is derived from apreviously identified RNAi molecule (i.e., the reference RNAi molecule)can be designed by introducing one or more internal, non-nucleotidespacers portions within the guide strand of the reference RNAi molecule.In another embodiment, the single-stranded RNAi molecules can bedesigned de novo (i.e., not based on a known RNAi agent) for the purposeof knocking down expression of a particular target sequence.

The RNAi activity of a given single-stranded RNAi molecule of theinvention can be measured using known methods, such as those describedgenerally in Fire et al., PCT Publication No. WO99/32619, and asdescribed in the Examples section infra. In some embodiments, theinstant specification provides methods for selecting more efficacioussingle-stranded RNAi molecule designs by using one or more reporter geneconstructs comprising a constitutive promoter, such as a cytomegalovirus(CMV) or phosphoglycerate kinase (PGK) promoter, operably fused to, andcapable of altering the expression of one or more reporter genes, suchas a luciferase, chloramphenicol (CAT), or β-galactosidase, which, inturn, is operably fused in-frame to a portion of the target sequencethat is whole or partially complementary to the ssRNAi to be tested.These reporter gene expression constructs may be co-transfected with oneor more ssRNAi molecules and a control (e.g., corresponding miRNAmimetic that does not contain the internal non-nucleotide spacer). Thecapacity of a given ssRNAi molecule to mediate RNAi of a target mRNA maybe determined by comparing the measured reporter gene activity in cellstransfected with the ssRNAi molecule and the activity in cellstransfected with a negative control (i.e., in cells not transfected withthe ssRNAi molecule) and a positive control (e.g., in cells transfectedwith the corresponding miRNA mimetic that does not contain the internalnon-nucleotide spacer). The ssRNAi molecules having at least 20% ormore, preferably at least 40% or more, or 60% or more, or 80% or more,of the activity of their corresponding RNAi molecule, for example, thatdo not contain internal non-nucleotide spacers, are selected.

A person of skill in the art can screen single-stranded RNAi moleculesof this disclosure containing various non-nucleotide spacers todetermine which of molecules possess improved properties (e.g.,pharmacokinetic profile, bioavailability, stability) while maintainingthe ability to mediate RNAi in, for example, an animal model asdescribed herein or generally known in the art. Similarly, a person ofskill in the art can also screen single-stranded RNAi molecules of thisdisclosure having various conjugates to determine which of the RNAimolecule-conjugate complexes possess improved properties whilemaintaining the ability to mediate RNAi.

F. Compositions and Methods of Use

As set forth herein, single-stranded RNA molecules of the invention areRNAi agents preferably capable of participating in the cellular RNAipathway or otherwise capable of modulating the same or relatedpathway(s) and resulting in the inhibition of a target gene associatedwith a pathological or diseased condition. In the case of asingle-stranded RNA molecule that represents a miRNA mimetic, the ssRNAimolecule is designed to supplement or take the place of a corresponding,naturally-occurring miRNA, the reduced or otherwise unsuitably lowlevels of which have been associated with pathological or diseasedconditions. The single-stranded RNAi molecules of the invention thus areuseful reagents, which can be used in methods for a variety oftherapeutic, diagnostic, target validation, genomic discovery, geneticengineering, and pharmacogenomic applications.

A single-stranded RNA molecule of the invention can be introduced to acell, tissue, organism, in vitro system, or in vivo system to mediateRNAi against a target sequence. That target sequence may be anendogenous target gene or sequence. In one embodiment, thesingle-stranded RNAi molecules of the invention can be used for treatingorganisms having a disease characterized by the undesired production ofa protein.

In the case of a single-stranded RNAi molecule of the invention that isa miRNA mimetic, the target sequence is the target of a corresponding,naturally-occurring miRNA. In such a case, the single-stranded miRNAmimetic may regulate a number of genes, for example, downstream from itsmRNA target, whose expression levels are associated with or otherwiseregulated by the corresponding, naturally-occurring miRNA. Becauseaberrant expression levels of certain naturally-occurring miRNAs havebeen implicated in various human ailments, including, but not limitedto, hyperproliferative, angiogenic, or inflammatory diseases, states, oradverse conditions, the single-stranded miRNA mimetics of the presentinvention can offer valuable therapeutic opportunities. In this context,a single-stranded miRNA mimetic of this disclosure can regulate (e.g.,knockdown or up-regulate) expression of one or more downstream genes ofits corresponding endogenous miRNA, such that prevention, alleviation,or reduction of the severity or recurrence of one or more associateddisease symptoms can be achieved. Alternatively, for various distinctdisease models in which expression of one or more target mRNAs are notnecessarily reduced or at a lower-than-normal level as a consequence ofdiseases or other adverse conditions, introducing exogenous miRNAmimetics, such as one or more single-stranded miRNA mimetics of theinvention, may nonetheless result in a therapeutic result by affectingthe expression levels of genes associated with the disease pathway.

A single-stranded RNAi molecule of invention can also act similar to asiRNA molecule in targeting the coding region of a target gene,inhibiting the expression that gene and, thus, reducing proteinproduction. The protein that would have been produced if not forintroduction of the single-stranded RNAi molecule may be associated witha pathological or diseased condition (e.g., cancer).

In accordance with this disclosure herein, a single-stranded RNAimolecule of the invention, compositions thereof, and methods forinhibiting expression of one or more corresponding target mRNAs in acell or organism are provided. This disclosure provides methods andsingle-stranded RNAi molecule compositions for treating a subject,including a human cell, tissue or individual.

(i) Pharmaceutical Compositions and Formulations

The present disclosure includes single-stranded RNAi moleculecompositions prepared for storage or administration that include apharmaceutically effective amount of a desired RNAi molecule in apharmaceutically acceptable carrier or diluent. The single-stranded RNAimolecule compositions of the disclosure can be effectively employed aspharmaceutically-acceptable formulations. Pharmaceutically-acceptableformulations prevent, alter the occurrence or severity of, or treat(alleviate one or more symptom(s) to a detectable or measurable extent)a disease state or other adverse condition in a subject. Thus, apharmaceutical composition or formulation refers to a composition orformulation in a form suitable for administration into a cell, or asubject such as a human (e.g., systemic administration). Thepharmaceutical compositions of the present disclosure are formulated toallow the single-stranded RNAi molecule(s) contained therein to bebioavailable upon administration to a subject.

In certain embodiments, pharmaceutical compositions of this disclosurecan optionally include preservatives, antioxidants, stabilizers, dyes,flavoring agents, or any combination thereof. Exemplary preservativesinclude sodium benzoate, esters of p-hydroxybenzoic acid, and sorbicacid. A pharmaceutically acceptable formulation includes salts of theabove compounds, for example, acid addition salts, such as salts ofhydrochloric acid, hydrobromic acid, acetic acid, or benzene sulfonicacid. Acceptable carriers or diluents for therapeutic use are well knownin the pharmaceutical art, and are described, for example, inRemington's Pharmaceutical Sciences, Mack Publishing Co., A. R. Gennaroedit., 21st Edition, 2005.

In certain embodiments, aqueous suspensions containing one or moresingle-stranded RNAi molecules of the invention can be prepared in anadmixture with suitable excipients, such as suspending agents ordispersing or wetting agents. Exemplary suspending agents include sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia.Representative dispersing or wetting agents include naturally-occurringphosphatides (e.g., lecithin), condensation products of an alkyleneoxide with fatty acids (e.g., polyoxyethylene stearate), condensationproducts of ethylene oxide with long chain aliphatic alcohols (e.g.,heptadecaethyleneoxycetanol), condensation products of ethylene oxidewith partial esters derived from fatty acids and hexitol (e.g.,polyoxyethylene sorbitol monooleate), or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides (e.g., polyethylene sorbitan monooleate). In certainembodiments, the aqueous suspensions can optionally contain one or morepreservatives (e.g., ethyl or w-propyl-p-hydroxybenzoate), one or morecoloring agents, one or more flavoring agents, or one or more sweeteningagents (e.g., sucrose, saccharin). In additional embodiments,dispersible powders and granules suitable for preparation of an aqueoussuspension comprising one or more single-stranded RNAi molecules of theinvention can be prepared by the addition of water with thesingle-stranded RNAi molecules in admixture with a dispersing or wettingagent, suspending agent and optionally one or more preservative,coloring agent, flavoring agent, or sweetening agent.

In further embodiments, a single-stranded RNAi molecule of thisdisclosure can be formulated as oily suspensions or emulsions (e.g.,oil-in-water) by suspending the ssRNAi in, for example, a vegetable oil(e.g., arachis oil, olive oil, sesame oil or coconut oil) or a mineraloil (e.g., liquid paraffin). Suitable emulsifying agents can benaturally-occurring gums (e.g., gum acacia or gum tragacanth),naturally-occurring phosphatides (e.g., soy bean, lecithin, esters orpartial esters derived from fatty acids and hexitol), anhydrides (e.g.,sorbitan monooleate), or condensation products of partial esters withethylene oxide (e.g., polyoxyethylene sorbitan monooleate). In certainembodiments, the oily suspensions or emulsions can optionally contain athickening agent, such as beeswax, hard paraffin or cetyl alcohol. Inrelated embodiments, sweetening agents and flavoring agents canoptionally be added to provide palatable oral preparations. In yet otherembodiments, these compositions can be preserved by the optionallyadding an anti-oxidant, such as ascorbic acid.

In further embodiments, single-stranded RNAi molecules can be formulatedas syrups and elixirs with sweetening agents (e.g., glycerol, propyleneglycol, sorbitol, glucose or sucrose). Such formulations can alsocontain a demulcent, preservative, flavoring, coloring agent, or anycombination thereof.

In other embodiments, pharmaceutical compositions comprising asingle-stranded RNAi molecule of the invention can be in the form of asterile, injectable aqueous or oleaginous suspension. The sterile,injectable preparation can also be a sterile, injectable solution orsuspension in a non-toxic, parenterally-acceptable diluent or solvent(e.g., as a solution in 1,3-butanediol). Among the exemplary acceptablevehicles and solvents useful in the compositions of this disclosure iswater, Ringer's solution, or isotonic sodium chloride solution. Inaddition, sterile, fixed oils may be employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil can be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid find use in the preparation of parenteral formulations.

The single-stranded RNAi molecules of the invention can be administereddirectly, or can be complexed, for example, with cationic lipids orpackaged within liposomes, or otherwise delivered to target cells ortissues. Methods for the delivery of nucleic acid molecules aredescribed in Akhtar et al., 1992, Trends Cell Bio., 2:139; DeliveryStrategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995;Maurer et al., 1999, Mol. Membr. Biol. 16:129-140; Hofland and Huang,1999, Handb. Exp. Pharmacol. 137:165-192; and Lee et al., 2000, ACSSymp. Ser. 752:184-192. Beigelman et al., U.S. Pat. No. 6,395,713 andSullivan et al., PCT WO 94/02595 further describe the general methodsfor delivery of nucleic acid molecules. These protocols can be utilizedfor the delivery of virtually any nucleic acid molecule. Nucleic acidmolecules can be administered to cells by a variety of methods known tothose of skill in the art, including, but not restricted to,encapsulation in liposomes, by iontophoresis, or by incorporation intoother vehicles, such as biodegradable polymers, hydrogels, cyclodextrins(see for example, Gonzalez et al., 1999, Bioconjugate Chem., 10,1068-1074; Wang et al., International PCT Publication Nos. WO 03/47518and WO 03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCAmicrospheres (see for example U.S. Pat. No. 6,447,796 and US PatentApplication Publication No. US 2002130430), biodegradable nanocapsules,and bioadhesive microspheres, or by proteinaceous vectors (O'Hare andNormand, International PCT Publication No. WO 00/53722).

(ii) Carrier/Delivery Systems

In one aspect, the present invention provides carrier systems containingthe single-stranded RNAi molecules described herein. In someembodiments, the carrier system is a lipid-based carrier system,cationic lipid, or liposome nucleic acid complexes, a liposome, amicelle, a virosome, a lipid nanoparticle or a mixture thereof. In otherembodiments, the carrier system is a polymer-based carrier system suchas a cationic polymer-nucleic acid complex. In additional embodiments,the carrier system is a cyclodextrin-based carrier system such as acyclodextrin polymer-nucleic acid complex. In further embodiments, thecarrier system is a protein-based carrier system such as a cationicpeptide-nucleic acid complex. Preferably, the carrier system in a lipidnanoparticle (“LNP”) formulation.

In certain embodiments, the single-stranded RNAi molecules of theinvention are formulated with a lipid nanoparticle composition such asis described in U.S. patent application Ser. Nos. 11/353,630,11/586,102, 61/189,295, 61/204,878, 61/235,476, 61/249,807, and61/298,022. In certain preferred embodiments, the ssRNAi molecules ofthe invention are formulated with a lipid nanoparticle compositioncomprising a cationic lipid/Cholesterol/PEG-C-DMA/DSPC in a 40/48/2/10ratio or a cationic lipid/Cholesterol/PEG-DMG/DSPC in a 40/48/2/10ratio. In certain other embodiments, the invention features acomposition comprising a ssRNAi molecule of the invention formulatedwith any of the cationic lipid formulations described in U.S. PatentApplication Nos. 61/189,295, 61/204,878, 61/235,476, 61/249,807, and61/298,022.

Within certain embodiments of this disclosure, pharmaceuticalcompositions and methods are provided that feature the presence oradministration of one or more single-stranded RNAi molecule, combined,complexed, or conjugated with functional moiety, optionally formulatedwith a pharmaceutically-acceptable carrier, such as a diluent,stabilizer, buffer, or the like. Such conjugates and/or complexes can beused to facilitate delivery of RNAi molecules into a biological system,such as a cell. The conjugates and complexes provided by the instantinvention can impart therapeutic activity by transferring therapeuticcompounds across cellular membranes, altering the pharmacokinetics,and/or modulating the localization of nucleic acid molecules of theinvention. Non-limiting, examples of such conjugates are described inU.S. Publication Nos. US2008/0152661 A1 and US 2004/0162260 A1 (e.g.,CDM-LBA, CDM-Pip-LBA, CDM-PEG, CDM-NAG, etc.) and U.S. patentapplication Ser. Nos. 10/427,160 and 10/201,394; and U.S. Pat. Nos.6,528,631; 6,335,434; 6,235,886; 6,153,737; 5,214,136; and 5,138,045.

A single-stranded RNAi molecule of this disclosure can include aconjugate member on one or more of the nucleotides, at a terminal and/orinternal position(s), and/or on the spacer portion of the molecule. Theconjugate member can be, for example, a lipophile, a terpene, a proteinbinding agent, a vitamin, a carbohydrate, or a peptide. For example, theconjugate member can be naproxen, nitroindole (or another conjugate thatcontributes to stacking interactions), folate, ibuprofen, or a C5pyrimidine linker. In other embodiments, the conjugate member is aglyceride lipid conjugate (e.g., a dialkyl glyceride derivatives),vitamin E conjugates, or thio-cholesterols. In various embodiments,polyethylene glycol (PEG) can be covalently attached to single-strandedRNAi molecules of the present invention. The attached PEG can be anymolecular weight, preferably from about 100 to about 50,000 daltons(Da).

Within certain embodiments of this disclosure, pharmaceuticalcompositions and methods are provided that feature the presence oradministration of one or more single-stranded RNAi molecule, combined,complexed, or conjugated with a polypeptide or peptide, optionallyformulated with a pharmaceutically-acceptable carrier, such as adiluent, stabilizer, buffer, or the like. In certain embodiments, whenpeptide conjugate partners are used to enhance delivery of one or moresingle-stranded RNAi molecules of this disclosure into a target cell, orotherwise enhance stability or activity of the molecule when contactedwith a biological sample. Exemplary peptide conjugate members for usewithin these aspects of this disclosure, include peptides PN27, PN28,PN29, PN58, PN61, PN73, PN158, PN159, PN173, PN182, PN202, PN204, PN250,PN361, PN365, PN404, PN453, and PN509 as described, for example, in U.S.Patent Application Publication Nos. 2006/0040882 and 2006/0014289, andU.S. Provisional Patent Application No. 60/939,578, which are allincorporated herein by reference.

In one embodiment, this disclosure provides compositions suitable foradministering single-stranded RNAi molecules of this disclosure tospecific cell types, such as hepatocytes. For example, theasialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987, J. Biol. Chem.262:4429) is unique to hepatocytes and binds branched galactose-terminalglycoproteins, such as asialoorosomucoid (ASOR). Binding of suchglycoproteins or synthetic glycoconjugates to the receptor takes placewith an affinity that strongly depends on the degree of branching of theoligosaccharide chain, for example, triatennary structures are boundwith greater affinity than biatenarry or monoatennary chains (Baenzigerand Fiete, 1980, Cell 22: 611; Connolly et al., 1982, J. Biol. Chem.257:939). Lee and Lee (1987, Glycoconjugate J. 4:317) obtained this highspecificity through the use of N-acetyl-D-galactosamine as thecarbohydrate moiety, which has higher affinity for the receptor comparedto galactose. This “clustering effect” has also been described for thebinding and uptake of mannosyl-terminating glycoproteins orglycoconjugates (Ponpipom et al., 1981, J. Med. Chem. 24: 1388). The useof galactose and galactosamine based conjugates to transport exogenouscompounds across cell membranes can provide a targeted delivery approachto the treatment of liver disease. The use of bioconjugates can alsoprovide a reduction in the required dose of therapeutic compoundsrequired for treatment. Furthermore, therapeutic bioavailability,pharmacodynamics, and pharmacokinetic parameters can be modulatedthrough the use of bioconjugates of this disclosure.

In still another embodiment, a single-stranded RNAi molecule of theinvention may be conjugated to a polypeptide and admixed with one ormore non-cationic lipids or a combination of a non-cationic lipid and acationic lipid to form a composition that enhances intracellulardelivery of the RNAi molecule as compared to delivery resulting fromcontacting the target cells with a naked RNAi molecule without thelipids. In more detailed aspects of this disclosure, the mixture,complex or conjugate comprising a single-stranded RNAi molecule and apolypeptide can be optionally combined with (e.g., admixed or complexedwith) a cationic lipid, such as Lipofectine™. To produce thesecompositions comprised of a polypeptide, a single-stranded RNAi moleculeand a cationic lipid, the RNAi molecule and the polypeptide may be mixedtogether first in a suitable medium such as a cell culture medium, afterwhich the cationic lipid is added to the mixture to form an RNAimolecule/delivery peptide/cationic lipid composition. Optionally, thepeptide and cationic lipid can be mixed together first in a suitablemedium such as a cell culture medium, followed by the addition of thesingle-stranded RNAi molecule to form the RNAi molecule/deliverypeptide/cationic lipid composition.

This disclosure also features the use of single-stranded RNAi moleculecompositions comprising surface-modified liposomes containingpoly(ethylene glycol) lipids (PEG-modified, or long-circulatingliposomes or stealth liposomes). These formulations may offer increasedaccumulation of drugs in target tissues (Lasic et al., 1995, Chem. Rev.95:2601; Ishiwata et al., 1995, Chem. Pharm. Bull. 43:1005). Suchliposomes have been shown to accumulate selectively in tumors,presumably by extravasation and capture in the neovascularized targettissues (Lasic et al., 1995, Science 267:1215; Oku et al., 1995,Biochim. Biophys. Acta 1238:86). The long-circulating liposomes enhancethe pharmacokinetics and pharmacodynamics of nucleic acid molecules ascompared to conventional cationic liposomes, which are known toaccumulate in tissues of the mononuclear phagocytic system (MPS) (Liu etal., 1995, J. Biol. Chem. 42:24864; Choi et al., PCT Publication No. WO96/10391; Ansell et al., PCT Publication No. WO 96/10390; Holland etal., PCT Publication No. WO 96/10392). Long-circulating liposomes mayalso provide additional protection from nuclease degradation as comparedto cationic liposomes, in theory due to avoiding accumulation inmetabolically aggressive MPS tissues, such as the liver and spleen.

In some embodiments, the RNAi molecules of the invention can also beformulated or complexed with polyethyleneimine and derivatives thereof,such as polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine(PEI-PEG-GAL) orpolyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine(PEI-PEG-triGAL) derivatives. In one embodiment, the nucleic acidmolecules of the invention are formulated as described in U.S. PatentApplication Publication No. 20030077829.

In other embodiments, single-stranded RNAi molecules of the inventionare complexed with membrane disruptive agents such as those described inU.S. Patent Application Publication No. 20010007666. In still otherembodiments, the membrane disruptive agent or agents and the RNAimolecule are also complexed with a cationic lipid or helper lipidmolecule, such as those lipids described in U.S. Pat. No. 6,235,310.

In certain embodiments, single-stranded RNAi molecules of the inventionare complexed with delivery systems as described in U.S. PatentApplication Publication Nos. 2003077829; 20050287551; 20050164220;20050191627; 20050118594; 20050153919; 20050085486; and 20030158133; andInternational PCT Publication Nos. WO 00/03683 and WO 02/087541.

In some embodiments, a liposomal formulation of the invention comprisesa RNAi molecule of the invention formulated or complexed with compoundsand compositions described in U.S. Pat. Nos. 6,858,224; 6,534,484;6,287,591; 6,835,395; 6,586,410; 6,858,225; 6,815,432; 6,586,001;6,120,798; 6,977,223; 6,998,115; 5,981,501; 5,976,567; 5,705,385; andU.S. Patent Application Publication Nos. 2006/0019912; 2006/0019258;2006/0008909; 2005/0255153; 2005/0079212; 2005/0008689; 2003/0077829,2005/0064595, 2005/0175682, 2005/0118253; 2004/0071654; 2005/0244504;2005/0265961 and 2003/0077829.

The present disclosure also features a method for preparingsingle-stranded RNAi molecule nanoparticles. A first solution containingmelamine derivatives is dissolved in an organic solvent such as dimethylsulfoxide, or dimethyl formamide to which an acid such as HCl has beenadded. The concentration of HCl would be about 3.3 moles of HCl forevery mole of the melamine derivative. The first solution is then mixedwith a second solution, which includes a nucleic acid dissolved orsuspended in a polar or hydrophilic solvent (e.g., an aqueous buffersolution containing, for instance, ethylenediaminetraacetic acid (EDTA),or tris(hydroxymethyl) aminomethane (TRIS), or combinations thereof. Themixture forms a first emulsion. The mixing can be done using anystandard technique such as, for example, sonication, vortexing, or in amicro fluidizer. The resultant nucleic acid particles can be purifiedand the organic solvent removed using size-exclusion chromatography ordialysis or both. The complexed nucleic acid nanoparticles can then bemixed with an aqueous solution containing either polyarginine or aGln-Asn polymer, or both, in an aqueous solution. A preferred molecularweight of each polymer is about 5000 to about 15,000 Daltons. This formsa solution containing nanoparticles of nucleic acid complexed with themelamine derivative and the polyarginine and the Gln-Asn polymers. Themixing steps are carried out in a manner that minimizes shearing of thenucleic acid while producing nanoparticles on average smaller than about200 nanometers in diameter. It is believed that the polyargininecomplexes with the negative charge of the phosphate groups within theminor groove of the nucleic acid, and the polyarginine wraps around thetrimeric nucleic acid complex. At either terminus of the polyarginineother moieties, such as the TAT polypeptide, mannose or galactose, canbe covalently bound to the polymer to direct binding of the nucleic acidcomplex to specific tissues, such as to the liver when galactose isused. While not being bound to theory, it is believed that the Gln-Asnpolymer complexes with the nucleic acid complex within the major grooveof the nucleic acid through hydrogen bonding with the bases of thenucleic acid. The polyarginine and the Gln-Asn polymer should be presentat a concentration of 2 moles per every mole of nucleic acid having 20base pairs. The concentration should be increased proportionally for anucleic acid having more than 20 base pairs. For example, if the nucleicacid has 25 base pairs, the concentration of the polymers should be2.5-3 moles per mole of double-stranded nucleic acid. The resultantnanoparticles can be purified by standard means such as size exclusionchromatography followed by dialysis. The purified complexednanoparticles can then be lyophilized using techniques well known in theart. One embodiment of the present disclosure provides nanoparticlesless than 100 nanometers (nm) comprising a single-stranded RNAimolecule.

(iii) Treatment

Subjects (e.g., mammalian, human) amendable for treatment using thesingle-stranded RNAi molecules of the invention (optionally substitutedor modified or conjugated), compositions thereof, and methods of thepresent disclosure include those suffering from one or more disease orcondition mediated, at least in part, by an aberrant expression level ofthe target gene or sequence, those at risk of developing a diseasecaused by or associated with the aberrant levels of a targetgene/sequence, or those which are amenable to treatment by replenishingor increasing the level of RNAi mediated by the corresponding ssRNAimolecule, including a hyperproliferative (e.g., cancer), angiogenic,metabolic, or inflammatory (e.g., arthritis) disease or disorder orcondition.

Compositions and methods disclosed herein are useful in the treatment ofa wide variety of target viruses, including retrovirus, such as humanimmunodeficiency virus (HIV), Hepatitis C Virus, Hepatitis B Virus,Coronavirus, as well as respiratory viruses, including human RespiratorySyncytial Virus, human Metapneumovirus, human Parainfluenza virus,Rhinovirus and Influenza virus.

In other examples, the compositions and methods of this disclosure areuseful as therapeutic tools to treat or prevent symptoms of, forexample, hyperproliferative disorders. Exemplary hyperproliferativedisorders include neoplasms, carcinomas, sarcomas, tumors, or cancer.More exemplary hyperproliferative disorders include oral cancer, throatcancer, laryngeal cancer, esophageal cancer, pharyngeal cancer,nasopharyngeal cancer, oropharyngeal cancer, gastrointestinal tractcancer, gastrointestinal stromal tumors (GIST), small intestine cancer,colon cancer, rectal cancer, colorectal cancer, anal cancer, pancreaticcancer, breast cancer, cervical cancer, uterine cancer, vulvar cancer,vaginal cancer, urinary tract cancer, bladder cancer, kidney cancer,adrenocortical cancer, islet cell carcinoma, gallbladder cancer, stomachcancer, prostate cancer, ovarian cancer, endometrial cancer,trophoblastic tumor, testicular cancer, penial cancer, bone cancer,osteosarcoma, liver cancer, extrahepatic bile duct cancer, skin cancer,basal cell carcinoma (BCC), lung cancer, small cell lung cancer,non-small cell lung cancer (NSCLC), brain cancer, melanoma, Kaposi'ssarcoma, eye cancer, head and neck cancer, squamous cell carcinoma ofhead and neck, tymoma, thymic carcinoma, thyroid cancer, parathyroidcancer, Hippel-Lindau syndrome, leukemia, acute myeloid leukemia,chronic myelogenous leukemia, acute lymphoblastic leukemia, hairy cellleukemia, lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, T-celllymphoma, multiple myeloma, malignant pleural mesothelioma, Barrett'sadenocarcinoma, Wilm's tumor, or the like. In other examples, thecompositions and methods of this disclosure are useful as therapeutictools to regulate expression of one or more target gene to treat orprevent symptoms of, for example, inflammatory disorders. Exemplaryinflammatory disorders include diabetes mellitus, rheumatoid arthritis,pannus growth in inflamed synovial lining, collagen-induced arthritis,spondylarthritis, ankylosing spondylitis, multiple sclerosis,encephalomyelitis, inflammatory bowel disease, Chron's disease,psoriasis or psoriatic arthritis, myasthenia gravis, systemic lupuserythematosis, graft-versus-host disease, atherosclerosis, andallergies.

Other exemplary disorders that can be treated with single-stranded RNAimolecules, compositions and methods of the instant disclosure includemetabolic disorders, cardiac disease, pulmonary disease,neovascularization, ischemic disorders, age-related maculardegeneration, diabetic retinopathy, glomerulonephritis, diabetes,asthma, chronic obstructive pulmonary disease, chronic bronchitis,lymphangiogenesis, and atherosclerosis.

Within additional aspects, combination formulations and methods areprovided comprising an effective amount of one or more single-strandedRNAi molecules in combination with one or more secondary or adjunctiveactive agents that are formulated together or administered coordinatelywith the single-stranded RNAi molecules of the invention to control oneor more target gene-associated disease or condition as described herein.Useful adjunctive therapeutic agents in these combinatorial formulationsand coordinate treatment methods include, for example, enzymatic nucleicacid molecules, allosteric nucleic acid molecules, guide, decoy, oraptamer nucleic acid molecules, antibodies such as monoclonalantibodies, small molecules and other organic or inorganic compoundsincluding metals, salts and ions, and other drugs and active agentsindicated for treating one or more target gene-associated disease orcondition, including chemotherapeutic agents used to treat cancer,steroids, non-steroidal anti-inflammatory drugs (NSAIDs), or the like.Exemplary chemotherapeutic agents include alkylating agents (e.g.,cisplatin, oxaliplatin, carboplatin, busulfan, nitrosoureas, nitrogenmustards, uramustine, temozolomide), antimetabolites (e.g., aminopterin,methotrexate, mercaptopurine, fluorouracil, cytarabine), taxanes (e.g.,paclitaxel, docetaxel), anthracyclines (e.g., doxorubicin, daunorubicin,epirubicin, idaruicin, mitoxantrone, valrubicin), bleomycin, mytomycin,actinomycin, hydroxyurea, topoisomerase inhibitors (e.g., camptothecin,topotecan, irinotecan, etoposide, teniposide), monoclonal antibodies(e.g., alemtuzumab, bevacizumab, cetuximab, gemtuzumab, panitumumab,rituximab, tositumomab, trastuzumab), vinca alkaloids (e.g.,vincristine, vinblastine, vindesine, vinorelbine), cyclophosphamide,prednisone, leucovorin, oxaliplatin.

To practice the coordinate administration methods of this disclosure, asingle-stranded RNAi molecule is administered simultaneously orsequentially in a coordinated treatment protocol with one or moresecondary or adjunctive therapeutic agents described herein or known inthe art. The coordinate administration may be done in either order, andthere may be a time period while only one or both (or all) activetherapeutic agents, individually or collectively, exert their biologicalactivities. A distinguishing aspect of all such coordinate treatmentmethods is that the single-stranded RNAi molecule(s) present in acomposition elicits some favorable clinical response, which may or maynot be in conjunction with a secondary clinical response provided by thesecondary therapeutic agent. For example, the coordinate administrationof a single-stranded RNAi molecule with a secondary therapeutic agent ascontemplated herein can yield an enhanced (e.g., synergistic)therapeutic response beyond the therapeutic response elicited by eitheror both the purified single-stranded RNAi molecule and the secondarytherapeutic agent alone.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of subject being treated, the physicalcharacteristics of the specific subject under consideration fortreatment (e.g., age, body weight, general health, sex, diet),concurrent medication, rate of excretion, drug combination, the severityof the particular disease undergoing therapy, and other factors thatthose skilled in the medical arts will recognize. For example, an amountbetween about 0.1 mg/kg and about 140 mg/kg body weight/day of activeingredients may be administered depending on the potency of asingle-stranded RNAi molecule of this disclosure (about 0.5 mg to about7 g per patient per day). The amount of active ingredient that can becombined with the carrier materials to produce a single dosage formvaries depending upon the host treated and the particular mode ofadministration. Dosage unit forms generally contain between from about 1mg to about 500 mg of an active ingredient.

Nucleic acid molecules can be administered to cells or organisms by avariety of methods known to those of skill in the art, includingadministration of formulations that comprise a single-stranded RNAimolecule, or formulations that further comprise one or more additionalcomponents, such as a pharmaceutically acceptable carrier, diluent,excipient, adjuvant, emulsifier, buffer, stabilizer, preservative, orthe like. In certain embodiments, a single-stranded RNAi molecule of theinvention, and/or the polypeptide can be encapsulated in liposomes,administered by iontophoresis, or incorporated into other vehicles, suchas hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesivemicrospheres, or proteinaceous vectors (see, e.g., PCT Publication No.WO 00/53722). Alternatively, a nucleic acid/peptide/vehicle combinationcan be locally delivered by direct injection or by use of an infusionpump. Direct injection of the nucleic acid molecules of this disclosure,whether subcutaneous, intramuscular, or intradermal, can take placeusing standard needle and syringe methodologies, or by needle-freetechnologies, such as those described in Conroy et al, (1999, Clin.Cancer Res. 5:2330) and PCT Publication No. WO 99/31262.

The formulations of the present disclosure, having an amount of asingle-stranded RNAi molecule sufficient to treat or prevent a disorderassociated with target gene expression are, for example, suitable fortopical (e.g., creams, ointments, skin patches, eye drops, ear drops)application or administration. Other routes of administration includeoral, parenteral, sublingual, bladder washout, vaginal, rectal, enteric,suppository, nasal, and inhalation. The term “parenteral,” as usedherein, includes subcutaneous, intravenous, intramuscular,intraarterial, intraabdominal, intraperitoneal, intraarticular,intraocular or retrobulbar, intraaural, intrathecal, intracavitary,intracelial, intraspinal, intrapulmonary or transpulmonary,intrasynovial, and intraurethral injection or infusion techniques. Thecompositions of the present disclosure may also be formulated and usedas a tablet, capsule or elixir for oral administration, suppository forrectal administration, sterile solution, or suspension for injectableadministration, either with or without other compounds known in the art.For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

Further methods for delivery of nucleic acid molecules, such assingle-stranded RNAi molecules of this invention, have been describedin, for example, Boado et al., 1998, J. Pharm. Sci 87:1308; Tyler etal., 1999, FEBS Lett. 421:2m; Pardridge et al., 1995, Proc. Nat'l Acad.Sci. USA 92:5592; Boado, 1995, Adv. Drug Delivery Rev. 15:73;Aldrian-Herrada et al. 1998, Nucleic Acids Res. 26:4910; Tyler et al.,1999, Proc. Nat'l Acad. Sci. USA 96:7053; Akhtar et al., 1992, TrendsCell Bio. 2:139; “Delivery Strategies for Guide OligonucleotideTherapeutics,” ed. Akhtar, 1995, Maurer et al., 1999 Mol. Membr. Biol.16:129; Lee et al., 2000, ACS Symp. Ser. 752:184. In addition to in vivoand therapeutic applications, a skilled person in the art willappreciate that the single-stranded RNAi molecules of the presentdisclosure are useful in a wide variety of in vitro applications, suchas in scientific and commercial research (e.g., elucidation ofphysiological pathways, drug discovery and development), and medical andveterinary diagnostics.

All U.S. patents, U.S. patent publications, U.S. patent applications,foreign patents, foreign patent applications, non-patent publications,figures, and websites referred to in this specification are expresslyincorporated herein by reference, in their entirety.

Table 1 lists certain endogenous human miRNA sequences, wherein the seedsequences, confirmed or projected, are capitalized. All miRNA sequencesin Table 1 are shown in 5′ to 3′ orientation. Other miRNA sequences ofthe present invention may be found in the miRBase database, the contentof which is incorporated by reference herein.

TABLE 1 SEQ ID miRNA name miRBase number Sequence NO hsa-let-7aMIMAT0000062 UGAGGUAGuagguuguauaguu 1 hsa-1et-7a* MIMAT0004481CUAUACAAucuacugucuuuc 2 hsa-1et-7a-2* MIMAT0010195CUGUACAGccuccuagcuuucc 3 hsa-let-7b MIMAT0000063 UGAGGUAGuagguugugugguu4 hsa-1et-7b* MIMAT0004482 CUAUACAAccuacugccuuccc 5 hsa-let-7cMIMAT0000064 UGAGGUAGuagguuguaugguu 6 hsa-1et-7c* MIMAT0004483UAGAGUUAcacccugggaguua 7 hsa-let-7d MIMAT0000065 AGAGGUAGuagguugcauaguu8 hsa-1et-7d* MIMAT0004484 CUAUACGAccugcugccuuucu 9 hsa-let-7eMIMAT0000066 UGAGGUAGgagguuguauaguu 10 hsa-1et-7e* MIMAT0004485CUAUACGGccuccuagcuuucc 11 hsa-let-7f MIMAT0000067 UGAGGUAGuagauuguauaguu12 hsa-let-7f-1* MIMAT0004486 CUAUACAAucuauugccuuccc 13 hsa-1et-7f-2*MIMAT0004487 CUAUACAGucuacugucuuucc 14 hsa-miR-15a MIMAT0000068UAGCAGCAcauaaugguuugug 15 hsa-miR-15a* MIMAT0004488CAGGCCAUauugugcugccuca 16 hsa-miR-16 MIMAT0000069 UAGCAGCAcguaaauauuggcg17 hsa-miR-16-1* MIMAT0004489 CCAGUAUUaacugugcugcuga 18 hsa-miR-17MIMAT0000070 CAAAGUGCuuacagugcagguag 19 hsa-miR-17* MIMAT0000071ACUGCAGUgaaggcacuuguag 20 hsa-miR-18a MIMAT0000072UAAGGUGCaucuagugcagauag 21 hsa-miR-18a* MIMAT0002891ACUGCCCUaagugcuccuucugg 22 hsa-miR-19a* MIMAT0004490AGUUUUGCauaguugcacuaca 23 hsa-miR-19a MIMAT0000073UGUGCAAAucuaugcaaaacuga 24 hsa-miR-19b-1* MIMAT0004491AGUUUUGCagguuugcauccagc 25 hsa-miR-19b MIMAT0000074UGUGCAAAuccaugcaaaacuga 26 hsa-miR-19b-2* MIMAT0004492AGUUUUGCagguuugcauuuca 27 hsa-miR-20a MIMAT0000075UAAAGUGCuuauagugcagguag 28 hsa-miR-20a* MIMAT0004493ACUGCAUUaugagcacuuaaag 29 hsa-miR-21 MIMAT0000076 UAGCUUAUcagacugauguuga30 hsa-miR-21* MIMAT0004494 CAACACCAgucgaugggcugu 31 hsa-miR-22*MIMAT0004495 AGUUCUUCaguggcaagcuuua 32 hsa-miR-22 MIMAT0000077AAGCUGCCaguugaagaacugu 33 hsa-miR-23a* MIMAT0004496GGGGUUCCuggggaugggauuu 34 hsa-miR-23a MIMAT0000078 AUCACAUUgccagggauuucc35 hsa-miR-24-1* MIMAT0000079 UGCCUACUgagcugauaucagu 36 hsa-miR-24MIMAT0000080 UGGCUCAGuucagcaggaacag 37 hsa-miR-24-2* MIMAT0004497UGCCUACUgagcugaaacacag 38 hsa-miR-25* MIMAT0004498 AGGCGGAGacuugggcaauug39 hsa-miR-25 MIMAT0000081 CAUUGCACuugucucggucuga 40 hsa-miR-26aMIMAT0000082 UUCAAGUAauccaggauaggcu 41 hsa-miR-26a-1* MIMAT0004499CCUAUUCUugguuacuugcacg 42 hsa-miR-26b MIMAT0000083 UUCAAGUAauucaggauaggu43 hsa-miR-26b* MIMAT0004500 CCUGUUCUccauuacuuggcuc 44 hsa-miR-27a*MIMAT0004501 AGGGCUUAgcugcuugugagca 45 hsa-miR-27a MIMAT0000084UUCACAGUggcuaaguuccgc 46 hsa-miR-28-5p MIMAT0000085AAGGAGCUcacagucuauugag 47 hsa-miR-28-3p MIMAT0004502CACUAGAUugugagcuccugga 48 hsa-miR-29a* MIMAT0004503ACUGAUUUcuuuugguguucag 49 hsa-miR-29a MIMAT0000086UAGCACCAucugaaaucgguua 50 hsa-miR-30a MIMAT0000087UGUAAACAuccucgacuggaag 51 hsa-miR-30a* MIMAT0000088CUUUCAGUcggauguuugcagc 52 hsa-miR-31 MIMAT0000089 AGGCAAGAugcuggcauagcu53 hsa-miR-31* MIMAT0004504 UGCUAUGCcaacauauugccau 54 hsa-miR-32MIMAT0000090 UAUUGCACauuacuaaguugca 55 hsa-miR-32* MIMAT0004505CAAUUUAGugugugugauauuu 56 hsa-miR-33a MIMAT0000091 GUGCAUUGuaguugcauugca57 hsa-miR-33a* MIMAT0004506 CAAUGUUUccacagugcaucac 58 hsa-miR-92a-1*MIMAT0004507 AGGUUGGGaucgguugcaaugcu 59 hsa-miR-92a MIMAT0000092UAUUGCACuugucccggccugu 60 hsa-miR-92a-2* MIMAT0004508GGGUGGGGauuuguugcauuac 61 hsa-miR-93 MIMAT0000093CAAAGUGCuguucgugcagguag 62 hsa-miR-93* MIMAT0004509ACUGCUGAgcuagcacuucccg 63 hsa-miR-95 MIMAT0000094 UUCAACGGguauuuauugagca64 hsa-miR-96 MIMAT0000095 UUUGGCACuagcacauuuuugcu 65 hsa-miR-96*MIMAT0004510 AAUCAUGUgcagugccaauaug 66 hsa-miR-98 MIMAT0000096UGAGGUAGuaaguuguauuguu 67 hsa-miR-99a MIMAT0000097AACCCGUAgauccgaucuugug 68 hsa-miR-99a* MIMAT0004511CAAGCUCGcuucuaugggucug 69 hsa-miR-100 MIMAT0000098AACCCGUAgauccgaacuugug 70 hsa-miR-100* MIMAT0004512CAAGCUUGuaucuauagguaug 71 hsa-miR-101* MIMAT0004513CAGUUAUCacagugcugaugcu 72 hsa-miR-101 MIMAT0000099 UACAGUACugugauaacugaa73 hsa-miR-29b-1* MIMAT0004514 GCUGGUUUcauauggugguuuaga 74 hsa-miR-29bMIMAT0000100 UAGCACCAuuugaaaucaguguu 75 hsa-miR-29b-2* MIMAT0004515CUGGUUUCacaugguggcuuag 76 hsa-miR-103-2* MIMAT0009196AGCUUCUUuacagugcugccuug 77 hsa-miR-103 MIMAT0000101AGCAGCAUuguacagggcuauga 78 hsa-miR-105 MIMAT0000102UCAAAUGCucagacuccuguggu 79 hsa-miR-105* MIMAT0004516ACGGAUGUuugagcaugugcua 80 hsa-miR-106a MIMAT0000103AAAAGUGCuuacagugcagguag 81 hsa-miR-106a* MIMAT0004517CUGCAAUGuaagcacuucuuac 82 hsa-miR-107 MIMAT0000104AGCAGCAUuguacagggcuauca 83 hsa-miR-16-2* MIMAT0004518CCAAUAUUacugugcugcuuua 84 hsa-miR-192 MIMAT0000222 CUGACCUAugaauugacagcc85 hsa-miR-192* MIMAT0004543 CUGCCAAUuccauaggucacag 86 hsa-miR-196aMIMAT0000226 UAGGUAGUuucauguuguuggg 87 hsa-miR-197 MIMAT0000227UUCACCACcuucuccacccagc 88 hsa-miR-198 MIMAT0000228GGUCCAGAggggagauagguuc 89 hsa-miR-199a-5p MIMAT0000231CCCAGUGUucagacuaccuguuc 90 hsa-miR-199a-3p MIMAT0000232ACAGUAGUcugcacauugguua 91 hsa-miR-208a MIMAT0000241AUAAGACGagcaaaaagcuugu 92 hsa-miR-129-5p MIMAT0000242CUUUUUGCggucugggcuugc 93 hsa-miR-129* MIMAT0004548AAGCCCUUaccccaaaaaguau 94 hsa-miR-148a* MIMAT0004549AAAGUUCUgagacacuccgacu 95 hsa-miR-148a MIMAT0000243UCAGUGCAcuacagaacuuugu 96 hsa-miR-30c MIMAT0000244UGUAAACAuccuacacucucagc 97 hsa-miR-30c-2* MIMAT0004550CUGGGAGAaggcuguuuacucu 98 hsa-miR-30d MIMAT0000245UGUAAACAuccccgacuggaag 99 hsa-miR-30d* MIMAT0004551CUUUCAGUcagauguuugcugc 100 hsa-miR-139-5p MIMAT0000250UCUACAGUgcacgugucuccag 101 hsa-miR-139-3p MIMAT0004552GGAGACGCggcccuguuggagu 102 hsa-miR-147 MIMAT0000251 GUGUGUGGaaaugcuucugc103 hsa-miR-7 MIMAT0000252 UGGAAGACuagugauuuuguugu 104 hsa-miR-7-1*MIMAT0004553 CAACAAAUcacagucugccaua 105 hsa-miR-7-2* MIMAT0004554CAACAAAUcccagucuaccuaa 106 hsa-miR-10a MIMAT0000253UACCCUGUagauccgaauuugug 107 hsa-miR-10a* MIMAT0004555CAAAUUCGuaucuaggggaaua 108 hsa-miR-10b MIMAT0000254UACCCUGUagaaccgaauuugug 109 hsa-miR-10b* MIMAT0004556ACAGAUUCgauucuaggggaau 110 hsa-miR-34a MIMAT0000255UGGCAGUGucuuagcugguugu 111 hsa-miR-34a* MIMAT0004557CAAUCAGCaaguauacugcccu 112 hsa-miR-181a MIMAT0000256AACAUUCAacgcugucggugagu 113 hsa-miR-181a2* MIMAT0004558ACCACUGAccguugacuguacc 114 hsa-miR-181b MIMAT0000257AACAUUCAuugcugucggugggu 115 hsa-miR-181c MIMAT0000258AACAUUCAaccugucggugagu 116 hsa-miR-181c* MIMAT0004559AACCAUCGaccguugaguggac 117 hsa-miR-182 MIMAT0000259UUUGGCAAugguagaacucacacu 118 hsa-miR-182* MIMAT0000260UGGUUCUAgacuugccaacua 119 hsa-miR-183 MIMAT0000261UAUGGCACugguagaauucacu 120 hsa-miR-183* MIMAT0004560GUGAAUUAccgaagggccauaa 121 hsa-miR-187* MIMAT0004561GGCUACAAcacaggacccgggc 122 hsa-miR-187 MIMAT0000262UCGUGUCUuguguugcagccgg 123 hsa-miR-196a* MIMAT0004562CGGCAACAagaaacugccugag 124 hsa-miR-199b-5p MIMAT0000263CCCAGUGUuuagacuaucuguuc 125 hsa-miR-199b-3p MIMAT0004563ACAGUAGUcugcacauugguua 91 hsa-miR-203 MIMAT0000264GUGAAAUGuuuaggaccacuag 126 hsa-miR-204 MIMAT0000265UUCCCUUUgucauccuaugccu 127 hsa-miR-205 MIMAT0000266UCCUUCAUuccaccggagucug 128 hsa-miR-205* MIMAT0009197GAUUUCAGuggagugaaguuc 129 hsa-miR-210 MIMAT0000267CUGUGCGUgugacagcggcuga 130 hsa-miR-211 MIMAT0000268UUCCCUUUgucauccuucgccu 131 hsa-miR-212 MIMAT0000269UAACAGUCuccagucacggcc 132 hsa-miR-181a* MIMAT0000270ACCAUCGAccguugauuguacc 133 hsa-miR-214* MIMAT0004564UGCCUGUCuacacuugcugugc 134 hsa-miR-214 MIMAT0000271ACAGCAGGcacagacaggcagu 135 hsa-miR-215 MIMAT0000272AUGACCUAugaauugacagac 136 hsa-miR-216a MIMAT0000273UAAUCUCAgcuggcaacuguga 137 hsa-miR-217 MIMAT0000274UACUGCAUcaggaacugauugga 138 hsa-miR-218 MIMAT0000275UUGUGCUUgaucuaaccaugu 139 hsa-miR-218-1* MIMAT0004565AUGGUUCCgucaagcaccaugg 140 hsa-miR-218-2* MIMAT0004566CAUGGUUCugucaagcaccgcg 141 hsa-miR-219-5p MIMAT0000276UGAUUGUCcaaacgcaauucu 142 hsa-miR-219-1-3p MIMAT0004567AGAGUUGAgucuggacgucccg 143 hsa-miR-220a MIMAT0000277CCACACCGuaucugacacuuu 144 hsa-miR-221* MIMAT0004568ACCUGGCAuacaauguagauuu 145 hsa-miR-221 MIMAT0000278AGCUACAUugucugcuggguuuc 146 hsa-miR-222* MIMAT0004569CUCAGUAGccaguguagauccu 147 hsa-miR-222 MIMAT0000279AGCUACAUcuggcuacugggu 148 hsa-miR-223* MIMAT0004570CGUGUAUUugacaagcugaguu 149 hsa-miR-223 MIMAT0000280UGUCAGUUugucaaauacccca 150 hsa-miR-224 MIMAT0000281CAAGUCACuagugguuccguu 151 hsa-miR-224* MIMAT0009198AAAAUGGUgcccuagugacuaca 152 hsa-miR-200b* MIMAT0004571CAUCUUACugggcagcauugga 153 hsa-miR-200b MIMAT0000318UAAUACUGccugguaaugauga 154 hsa-let-7g MIMAT0000414UGAGGUAGuaguuuguacaguu 155 hsa-1et-7g* MIMAT0004584CUGUACAGgccacugccuugc 156 hsa-let-7i MIMAT0000415 UGAGGUAGuaguuugugcuguu157 hsa-1et-7i* MIMAT0004585 CUGCGCAAgcuacugccuugcu 158 hsa-miR-1MIMAT0000416 UGGAAUGUaaagaaguauguau 159 hsa-miR-15b MIMAT0000417UAGCAGCAcaucaugguuuaca 160 hsa-miR-15b* MIMAT0004586CGAAUCAUuauuugcugcucua 161 hsa-miR-23b* MIMAT0004587UGGGUUCCuggcaugcugauuu 162 hsa-miR-23b MIMAT0000418AUCACAUUgccagggauuacc 163 hsa-miR-27b* MIMAT0004588AGAGCUUAgcugauuggugaac 164 hsa-miR-27b MIMAT0000419UUCACAGUggcuaaguucugc 165 hsa-miR-30b MIMAT0000420UGUAAACAuccuacacucagcu 166 hsa-miR-30b* MIMAT0004589CUGGGAGGuggauguuuacuuc 167 hsa-miR-122 MIMAT0000421UGGAGUGUgacaaugguguuug 168 hsa-miR-122* MIMAT0004590AACGCCAUuaucacacuaaaua 169 hsa-miR-124* MIMAT0004591CGUGUUCAcagcggaccuugau 170 hsa-miR-124 MIMAT0000422 UAAGGCACgcggugaaugcc171 hsa-miR-125b MIMAT0000423 UCCCUGAGacccuaacuuguga 172 hsa-miR-125b-1*MIMAT0004592 ACGGGUUAggcucuugggagcu 173 hsa-miR-128 MIMAT0000424UCACAGUGaaccggucucuuu 174 hsa-miR-130a* MIMAT0004593UUCACAUUgugcuacugucugc 175 hsa-miR-130a MIMAT0000425CAGUGCAAuguuaaaagggcau 176 hsa-miR-132* MIMAT0004594ACCGUGGCuuucgauuguuacu 177 hsa-miR-132 MIMAT0000426UAACAGUCuacagccauggucg 178 hsa-miR-133a MIMAT0000427UUUGGUCCccuucaaccagcug 179 hsa-miR-135a MIMAT0000428UAUGGCUUuuuauuccuauguga 180 hsa-miR-135a* MIMAT0004595UAUAGGGAuuggagccguggcg 181 hsa-miR-137 MIMAT0000429UUAUUGCUuaagaauacgcguag 182 hsa-miR-138 MIMAT0000430AGCUGGUGuugugaaucaggccg 183 hsa-miR-138-2* MIMAT0004596GCUAUUUCacgacaccaggguu 184 hsa-miR-140-5p MIMAT0000431CAGUGGUUuuacccuaugguag 185 hsa-miR-140-3p MIMAT0004597UACCACAGgguagaaccacgg 186 hsa-miR-141* MIMAT0004598CAUCUUCCaguacaguguugga 187 hsa-miR-141 MIMAT0000432UAACACUGucugguaaagaugg 188 hsa-miR-142-5p MIMAT0000433CAUAAAGUagaaagcacuacu 189 hsa-miR-142-3p MIMAT0000434UGUAGUGUuuccuacuuuaugga 190 hsa-miR-143* MIMAT0004599GGUGCAGUgcugcaucucuggu 191 hsa-miR-143 MIMAT0000435UGAGAUGAagcacuguagcuc 192 hsa-miR-144* MIMAT0004600GGAUAUCAucauauacuguaag 193 hsa-miR-144 MIMAT0000436 UACAGUAUagaugauguacu194 hsa-miR-145 MIMAT0000437 GUCCAGUUuucccaggaaucccu 195 hsa-miR-145*MIMAT0004601 GGAUUCCUggaaauacuguucu 196 hsa-miR-152 MIMAT0000438UCAGUGCAugacagaacuugg 197 hsa-miR-153 MIMAT0000439UUGCAUAGucacaaaagugauc 198 hsa-miR-191 MIMAT0000440CAACGGAAucccaaaagcagcug 199 hsa-miR-191* MIMAT0001618GCUGCGCUuggauuucgucccc 200 hsa-miR-9 MIMAT0000441UCUUUGGUuaucuagcuguauga 201 hsa-miR-9* MIMAT0000442AUAAAGCUagauaaccgaaagu 202 hsa-miR-125a-5p MIMAT0000443UCCCUGAGacccuuuaaccuguga 203 hsa-miR-125a-3p MIMAT0004602ACAGGUGAgguucuugggagcc 204 hsa-miR-125b-2* MIMAT0004603UCACAAGUcaggcucuugggac 205 hsa-miR-126* MIMAT0000444CAUUAUUAcuuuugguacgcg 206 hsa-miR-126 MIMAT0000445UCGUACCGugaguaauaaugcg 207 hsa-miR-127-5p MIMAT0004604CUGAAGCUcagagggcucugau 208 hsa-miR-127-3p MIMAT0000446UCGGAUCCgucugagcuuggcu 209 hsa-miR-129-3p MIMAT0004605AAGCCCUUaccccaaaaagcau 210 hsa-miR-134 MIMAT0000447UGUGACUGguugaccagagggg 211 hsa-miR-136 MIMAT0000448ACUCCAUUuguuuugaugaugga 212 hsa-miR-136* MIMAT0004606CAUCAUCGucucaaaugagucu 213 hsa-miR-138-1* MIMAT0004607GCUACUUCacaacaccagggcc 214 hsa-miR-146a MIMAT0000449UGAGAACUgaauuccauggguu 215 hsa-miR-146a* MIMAT0004608CCUCUGAAauucaguucuucag 216 hsa-miR-149 MIMAT0000450UCUGGCUCcgugucuucacuccc 217 hsa-miR-149* MIMAT0004609AGGGAGGGacgggggcugugc 218 hsa-miR-150 MIMAT0000451UCUCCCAAcccuuguaccagug 219 hsa-miR-150* MIMAT0004610CUGGUACAggccugggggacag 220 hsa-miR-154 MIMAT0000452UAGGUUAUccguguugccuucg 221 hsa-miR-154* MIMAT0000453AAUCAUACacgguugaccuauu 222 hsa-miR-184 MIMAT0000454UGGACGGAgaacugauaagggu 223 hsa-miR-185 MIMAT0000455UGGAGAGAaaggcaguuccuga 224 hsa-miR-185* MIMAT0004611AGGGGCUGgcuuuccucugguc 225 hsa-miR-186 MIMAT0000456CAAAGAAUucuccuuuugggcu 226 hsa-miR-186* MIMAT0004612GCCCAAAGgugaauuuuuuggg 227 hsa-miR-188-5p MIMAT0000457CAUCCCUUgcaugguggaggg 228 hsa-miR-188-3p MIMAT0004613CUCCCACAugcaggguuugca 229 hsa-miR-190 MIMAT0000458UGAUAUGUuugauauauuaggu 230 hsa-miR-193a-5p MIMAT0004614UGGGUCUUugcgggcgagauga 231 hsa-miR-193a-3p MIMAT0000459AACUGGCCuacaaagucccagu 232 hsa-miR-194 MIMAT0000460UGUAACAGcaacuccaugugga 233 hsa-miR-195 MIMAT0000461UAGCAGCAcagaaauauuggc 234 hsa-miR-195* MIMAT0004615CCAAUAUUggcugugcugcucc 235 hsa-miR-206 MIMAT0000462UGGAAUGUaaggaagugugugg 236 hsa-miR-320a MIMAT0000510AAAAGCUGgguugagagggcga 237 hsa-miR-200c* MIMAT0004657CGUCUUACccagcaguguuugg 238 hsa-miR-200c MIMAT0000617UAAUACUGccggguaaugaugga 239 hsa-miR-155 MIMAT0000646UUAAUGCUaaucgugauaggggu 240 hsa-miR-155* MIMAT0004658CUCCUACAuauuagcauuaaca 241 hsa-miR-194* MIMAT0004671CCAGUGGGgcugcuguuaucug 242 hsa-miR-106b MIMAT0000680UAAAGUGCugacagugcagau 243 hsa-miR-106b* MIMAT0004672CCGCACUGuggguacuugcugc 244 hsa-miR-29c* MIMAT0004673UGACCGAUuucuccugguguuc 245 hsa-miR-29c MIMAT0000681UAGCACCAuuugaaaucgguua 246 hsa-miR-30c-1* MIMAT0004674CUGGGAGAggguuguuuacucc 247 hsa-miR-200a* MIMAT0001620CAUCUUACcggacagugcugga 248 hsa-miR-200a MIMAT0000682UAACACUGucugguaacgaugu 249 hsa-miR-302a* MIMAT0000683ACUUAAACguggauguacuugcu 250 hsa-miR-302a MIMAT0000684UAAGUGCUuccauguuuugguga 251 hsa-miR-219-2-3p MIMAT0004675AGAAUUGUggcuggacaucugu 252 hsa-miR-34b* MIMAT0000685UAGGCAGUgucauuagcugauug 253 hsa-miR-34b MIMAT0004676CAAUCACUaacuccacugccau 254 hsa-miR-34c-5p MIMAT0000686AGGCAGUGuaguuagcugauugc 255 hsa-miR-34c-3p MIMAT0004677AAUCACUAaccacacggccagg 256 hsa-miR-299-5p MIMAT0002890UGGUUUACcgucccacauacau 257 hsa-miR-299-3p MIMAT0000687UAUGUGGGaugguaaaccgcuu 258 hsa-miR-301a MIMAT0000688CAGUGCAAuaguauugucaaagc 259 hsa-miR-99b MIMAT0000689CACCCGUAgaaccgaccuugcg 260 hsa-miR-99b* MIMAT0004678CAAGCUCGugucuguggguccg 261 hsa-miR-296-5p MIMAT0000690AGGGCCCCcccucaauccugu 262 hsa-miR-296-3p MIMAT0004679GAGGGUUGgguggaggcucucc 263 hsa-miR-130b* MIMAT0004680ACUCUUUCccuguugcacuac 264 hsa-miR-130b MIMAT0000691CAGUGCAAugaugaaagggcau 265 hsa-miR-30e MIMAT0000692UGUAAACAuccuugacuggaag 266 hsa-miR-30e* MIMAT0000693CUUUCAGUcggauguuuacagc 267 hsa-miR-26a-2* MIMAT0004681CCUAUUCUugauuacuuguuuc 268 hsa-miR-361-5p MIMAT0000703UUAUCAGAaucuccagggguac 269 hsa-miR-361-3p MIMAT0004682UCCCCCAGgugugauucugauuu 270 hsa-miR-362-5p MIMAT0000705AAUCCUUGgaaccuaggugugagu 271 hsa-miR-362-3p MIMAT0004683AACACACCuauucaaggauuca 272 hsa-miR-363* MIMAT0003385CGGGUGGAucacgaugcaauuu 273 hsa-miR-363 MIMAT0000707AAUUGCACgguauccaucugua 274 hsa-miR-365 MIMAT0000710UAAUGCCCcuaaaaauccuuau 275 hsa-miR-365* MIMAT0009199AGGGACUUucaggggcagcugu 276 hsa-miR-302b* MIMAT0000714ACUUUAACauggaagugcuuuc 277 hsa-miR-302b MIMAT0000715UAAGUGCUuccauguuuuaguag 278 hsa-miR-302c* MIMAT0000716UUUAACAUggggguaccugcug 279 hsa-miR-302c MIMAT0000717UAAGUGCUuccauguuucagugg 280 hsa-miR-302d* MIMAT0004685ACUUUAACauggaggcacuugc 281 hsa-miR-302d MIMAT0000718UAAGUGCUuccauguuugagugu 282 hsa-miR-367* MIMAT0004686ACUGUUGCuaauaugcaacucu 283 hsa-miR-367 MIMAT0000719AAUUGCACuuuagcaaugguga 284 hsa-miR-376c MIMAT0000720AACAUAGAggaaauuccacgu 285 hsa-miR-369-5p MIMAT0001621AGAUCGACcguguuauauucgc 286 hsa-miR-369-3p MIMAT0000721AAUAAUACaugguugaucuuu 287 hsa-miR-370 MIMAT0000722GCCUGCUGggguggaaccuggu 288 hsa-miR-371-5p MIMAT0004687ACUCAAACugugggggcacu 289 hsa-miR-371-3p MIMAT0000723AAGUGCCGccaucuuuugagugu 290 hsa-miR-372 MIMAT0000724AAAGUGCUgcgacauuugagcgu 291 hsa-miR-373* MIMAT0000725ACUCAAAAugggggcgcuuucc 292 hsa-miR-373 MIMAT0000726GAAGUGCUucgauuuuggggugu 293 hsa-miR-374a MIMAT0000727UUAUAAUAcaaccugauaagug 294 hsa-miR-374a* MIMAT0004688CUUAUCAGauuguauuguaauu 295 hsa-miR-375 MIMAT0000728UUUGUUCGuucggcucgcguga 296 hsa-miR-376a* MIMAT0003386GUAGAUUCuccuucuaugagua 297 hsa-miR-376a MIMAT0000729AUCAUAGAggaaaauccacgu 298 hsa-miR-377* MIMAT0004689AGAGGUUGcccuuggugaauuc 299 hsa-miR-377 MIMAT0000730AUCACACAaaggcaacuuuugu 300 hsa-miR-378* MIMAT0000731CUCCUGACuccagguccugugu 301 hsa-miR-378 MIMAT0000732ACUGGACUuggagucagaagg 302 hsa-miR-379 MIMAT0000733 UGGUAGACuauggaacguagg303 hsa-miR-379* MIMAT0004690 UAUGUAACaugguccacuaacu 304 hsa-miR-380*MIMAT0000734 UGGUUGACcauagaacaugcgc 305 hsa-miR-380 MIMAT0000735UAUGUAAUaugguccacaucuu 306 hsa-miR-381 MIMAT0000736UAUACAAGggcaagcucucugu 307 hsa-miR-382 MIMAT0000737GAAGUUGUucgugguggauucg 308 hsa-miR-383 MIMAT0000738AGAUCAGAaggugauuguggcu 309 hsa-miR-340 MIMAT0004692UUAUAAAGcaaugagacugauu 310 hsa-miR-340* MIMAT0000750UCCGUCUCaguuacuuuauagc 311 hsa-miR-330-5p MIMAT0004693UCUCUGGGccugugucuuaggc 312 hsa-miR-330-3p MIMAT0000751GCAAAGCAcacggccugcagaga 313 hsa-miR-328 MIMAT0000752CUGGCCCUcucugcccuuccgu 314 hsa-miR-342-5p MIMAT0004694AGGGGUGCuaucugugauuga 315 hsa-miR-342-3p MIMAT0000753UCUCACACagaaaucgcacccgu 316 hsa-miR-337-5p MIMAT0004695GAACGGCUucauacaggaguu 317 hsa-miR-337-3p MIMAT0000754CUCCUAUAugaugccuuucuuc 318 hsa-miR-323-5p MIMAT0004696AGGUGGUCcguggcgcguucgc 319 hsa-miR-323-3p MIMAT0000755CACAUUACacggucgaccucu 320 hsa-miR-326 MIMAT0000756 CCUCUGGGcccuuccuccag321 hsa-miR-151-5p MIMAT0004697 UCGAGGAGcucacagucuagu 322 hsa-miR-151-3pMIMAT0000757 CUAGACUGaagcuccuugagg 323 hsa-miR-135b MIMAT0000758UAUGGCUUuucauuccuauguga 324 hsa-miR-135b* MIMAT0004698AUGUAGGGcuaaaagccauggg 325 hsa-miR-148b* MIMAT0004699AAGUUCUGuuauacacucaggc 326 hsa-miR-148b MIMAT0000759UCAGUGCAucacagaacuuugu 327 hsa-miR-331-5p MIMAT0004700CUAGGUAUggucccagggaucc 328 hsa-miR-331-3p MIMAT0000760GCCCCUGGgccuauccuagaa 329 hsa-miR-324-5p MIMAT0000761CGCAUCCCcuagggcauuggugu 330 hsa-miR-324-3p MIMAT0000762ACUGCCCCaggugcugcugg 331 hsa-miR-338-5p MIMAT0004701AACAAUAUccuggugcugagug 332 hsa-miR-338-3p MIMAT0000763UCCAGCAUcagugauuuuguug 333 hsa-miR-339-5p MIMAT0000764UCCCUGUCcuccaggagcucacg 334 hsa-miR-339-3p MIMAT0004702UGAGCGCCucgacgacagagccg 335 hsa-miR-335 MIMAT0000765UCAAGAGCaauaacgaaaaaugu 336 hsa-miR-335* MIMAT0004703UUUUUCAUuauugcuccugacc 337 hsa-miR-133b MIMAT0000770UUUGGUCCccuucaaccagcua 338 hsa-miR-325 MIMAT0000771CCUAGUAGguguccaguaagugu 339 hsa-miR-345 MIMAT0000772GCUGACUCcuaguccagggcuc 340 hsa-miR-346 MIMAT0000773UGUCUGCCcgcaugccugccucu 341 hsa-miR-384 MIMAT0001075AUUCCUAGaaauuguucaua 342 hsa-miR-196b MIMAT0001080UAGGUAGUuuccuguuguuggg 343 hsa-miR-196b* MIMAT0009201UCGACAGCacgacacugccuuc 344 hsa-miR-422a MIMAT0001339ACUGGACUuagggucagaaggc 345 hsa-miR-423-5p MIMAT0004748UGAGGGGCagagagcgagacuuu 346 hsa-miR-423-3p MIMAT0001340AGCUCGGUcugaggccccucagu 347 hsa-miR-424 MIMAT0001341CAGCAGCAauucauguuuugaa 348 hsa-miR-424* MIMAT0004749CAAAACGUgaggcgcugcuau 349 hsa-miR-425 MIMAT0003393AAUGACACgaucacucccguuga 350 hsa-miR-425* MIMAT0001343AUCGGGAAugucguguccgccc 351 hsa-miR-18b MIMAT0001412UAAGGUGCaucuagugcaguuag 352 hsa-miR-18b* MIMAT0004751UGCCCUAAaugccccuucuggc 353 hsa-miR-20b MIMAT0001413CAAAGUGCucauagugcagguag 354 hsa-miR-20b* MIMAT0004752ACUGUAGUaugggcacuuccag 355 hsa-miR-448 MIMAT0001532UUGCAUAUguaggaugucccau 356 hsa-miR-429 MIMAT0001536UAAUACUGucugguaaaaccgu 357 hsa-miR-449a MIMAT0001541UGGCAGUGuauuguuagcuggu 358 hsa-miR-450a MIMAT0001545UUUUGCGAuguguuccuaauau 359 hsa-miR-431 MIMAT0001625UGUCUUGCaggccgucaugca 360 hsa-miR-431* MIMAT0004757CAGGUCGUcuugcagggcuucu 361 hsa-miR-433 MIMAT0001627AUCAUGAUgggcuccucggugu 362 hsa-miR-329 MIMAT0001629AACACACCugguuaaccucuuu 363 hsa-miR-451 MIMAT0001631AAACCGUUaccauuacugaguu 364 hsa-miR-452 MIMAT0001635AACUGUUUgcagaggaaacuga 365 hsa-miR-452* MIMAT0001636CUCAUCUGcaaagaaguaagug 366 hsa-miR-409-5p MIMAT0001638AGGUUACCcgagcaacuuugcau 367 hsa-miR-409-3p MIMAT0001639GAAUGUUGcucggugaaccccu 368 hsa-miR-412 MIMAT0002170ACUUCACCugguccacuagccgu 369 hsa-miR-410 MIMAT0002171AAUAUAACacagauggccugu 370 hsa-miR-376b MIMAT0002172AUCAUAGAggaaaauccauguu 371 hsa-miR-483-5p MIMAT0004761AAGACGGGaggaaagaagggag 372 hsa-miR-483-3p MIMAT0002173UCACUCCUcuccucccgucuu 373 hsa-miR-484 MIMAT0002174UCAGGCUCaguccccucccgau 374 hsa-miR-485-5p MIMAT0002175AGAGGCUGgccgugaugaauuc 375 hsa-miR-485-3p MIMAT0002176GUCAUACAcggcucuccucucu 376 hsa-miR-486-5p MIMAT0002177UCCUGUACugagcugccccgag 377 hsa-miR-486-3p MIMAT0004762CGGGGCAGcucaguacaggau 378 hsa-miR-487a MIMAT0002178AAUCAUACagggacauccaguu 379 hsa-miR-488* MIMAT0002804CCCAGAUAauggcacucucaa 380 hsa-miR-488 MIMAT0004763 UUGAAAGGcuauuucuugguc381 hsa-miR-489 MIMAT0002805 GUGACAUCacauauacggcagc 382 hsa-miR-490-5pMIMAT0004764 CCAUGGAUcuccaggugggu 383 hsa-miR-490-3p MIMAT0002806CAACCUGGaggacuccaugcug 384 hsa-miR-491-5p MIMAT0002807AGUGGGGAacccuuccaugagg 385 hsa-miR-491-3p MIMAT0004765CUUAUGCAagauucccuucuac 386 hsa-miR-511 MIMAT0002808GUGUCUUUugcucugcaguca 387 hsa-miR-146b-5p MIMAT0002809UGAGAACUgaauuccauaggcu 388 hsa-miR-146b-3p MIMAT0004766UGCCCUGUggacucaguucugg 389 hsa-miR-202* MIMAT0002810UUCCUAUGcauauacuucuuug 390 hsa-miR-202 MIMAT0002811 AGAGGUAUagggcaugggaa391 hsa-miR-492 MIMAT0002812 AGGACCUGcgggacaagauucuu 392 hsa-miR-493*MIMAT0002813 UUGUACAUgguaggcuuucauu 393 hsa-miR-493 MIMAT0003161UGAAGGUCuacugugugccagg 394 hsa-miR-432 MIMAT0002814UCUUGGAGuaggucauugggugg 395 hsa-miR-432* MIMAT0002815CUGGAUGGcuccuccaugucu 396 hsa-miR-494 MIMAT0002816UGAAACAUacacgggaaaccuc 397 hsa-miR-495 MIMAT0002817AAACAAACauggugcacuucuu 398 hsa-miR-496 MIMAT0002818UGAGUAUUacauggccaaucuc 399 hsa-miR-193b* MIMAT0004767CGGGGUUUugagggcgagauga 400 hsa-miR-193b MIMAT0002819AACUGGCCcucaaagucccgcu 401 hsa-miR-497 MIMAT0002820CAGCAGCAcacugugguuugu 402 hsa-miR-497* MIMAT0004768CAAACCACacugugguguuaga 403 hsa-miR-181d MIMAT0002821AACAUUCAuuguugucggugggu 404 hsa-miR-512-5p MIMAT0002822CACUCAGCcuugagggcacuuuc 405 hsa-miR-512-3p MIMAT0002823AAGUGCUGucauagcugagguc 406 hsa-miR-498 MIMAT0002824UUUCAAGCcagggggcguuuuuc 407 hsa-miR-520e MIMAT0002825AAAGUGCUuccuuuuugaggg 408 hsa-miR-515-5p MIMAT0002826UUCUCCAAaagaaagcacuuucug 409 hsa-miR-515-3p MIMAT0002827GAGUGCCUucuuuuggagcguu 410 hsa-miR-519e* MIMAT0002828UUCUCCAAaagggagcacuuuc 411 hsa-miR-519e MIMAT0002829AAGUGCCUccuuuuagaguguu 412 hsa-miR-520f MIMAT0002830AAGUGCUUccuuuuagaggguu 413 hsa-miR-519c-5p MIMAT0002831CUCUAGAGggaagcgcuuucug 414 hsa-miR-519c-3p MIMAT0002832AAAGUGCAucuuuuuagaggau 415 hsa-miR-520a-5p MIMAT0002833CUCCAGAGggaaguacuuucu 416 hsa-miR-520a-3p MIMAT0002834AAAGUGCUucccuuuggacugu 417 hsa-miR-526b MIMAT0002835CUCUUGAGggaagcacuuucugu 418 hsa-miR-526b* MIMAT0002836GAAAGUGCuuccuuuuagaggc 419 hsa-miR-519b-5p MIMAT0005454CUCUAGAGggaagcgcuuucug 414 hsa-miR-519b-3p MIMAT0002837AAAGUGCAuccuuuuagagguu 420 hsa-miR-525-5p MIMAT0002838CUCCAGAGggaugcacuuucu 421 hsa-miR-525-3p MIMAT0002839GAAGGCGCuucccuuuagagcg 422 hsa-miR-523* MIMAT0005449CUCUAGAGggaagcgcuuucug 414 hsa-miR-523 MIMAT0002840GAACGCGCuucccuauagagggu 423 hsa-miR-518f* MIMAT0002841CUCUAGAGggaagcacuuucuc 424 hsa-miR-518f MIMAT0002842GAAAGCGCuucucuuuagagg 425 hsa-miR-520b MIMAT0002843AAAGUGCUuccuuuuagaggg 426 hsa-miR-518b MIMAT0002844CAAAGCGCuccccuuuagaggu 427 hsa-miR-526a MIMAT0002845CUCUAGAGggaagcacuuucug 428 hsa-miR-520c-5p MIMAT0005455CUCUAGAGggaagcacuuucug 428 hsa-miR-520c-3p MIMAT0002846AAAGUGCUuccuuuuagagggu 429 hsa-miR-518c* MIMAT0002847UCUCUGGAgggaagcacuuucug 430 hsa-miR-518c MIMAT0002848CAAAGCGCuucucuuuagagugu 431 hsa-miR-524-5p MIMAT0002849CUACAAAGggaagcacuuucuc 432 hsa-miR-524-3p MIMAT0002850GAAGGCGCuucccuuuggagu 433 hsa-miR-517* MIMAT0002851CCUCUAGAuggaagcacugucu 434 hsa-miR-517a MIMAT0002852AUCGUGCAucccuuuagagugu 435 hsa-miR-519d MIMAT0002853CAAAGUGCcucccuuuagagug 436 hsa-miR-521 MIMAT0002854AACGCACUucccuuuagagugu 437 hsa-miR-520d-5p MIMAT0002855CUACAAAGggaagcccuuuc 438 hsa-miR-520d-3p MIMAT0002856AAAGUGCUucucuuuggugggu 439 hsa-miR-517b MIMAT0002857UCGUGCAUcccuuuagaguguu 440 hsa-miR-520g MIMAT0002858ACAAAGUGcuucccuuuagagugu 441 hsa-miR-516b MIMAT0002859AUCUGGAGguaagaagcacuuu 442 hsa-miR-516b* MIMAT0002860 UGCUUCCUuucagagggu443 hsa-miR-518e* MIMAT0005450 CUCUAGAGggaagcgcuuucug 414 hsa-miR-518eMIMAT0002861 AAAGCGCUucccuucagagug 444 hsa-miR-518a-5p MIMAT0005457CUGCAAAGggaagcccuuuc 445 hsa-miR-518a-3p MIMAT0002863GAAAGCGCuucccuuugcugga 446 hsa-miR-518d-5p MIMAT0005456CUCUAGAGggaagcacuuucug 428 hsa-miR-518d-3p MIMAT0002864CAAAGCGCuucccuuuggagc 447 hsa-miR-517c MIMAT0002866AUCGUGCAuccuuuuagagugu 448 hsa-miR-520h MIMAT0002867ACAAAGUGcuucccuuuagagu 449 hsa-miR-522* MIMAT0005451CUCUAGAGggaagcgcuuucug 414 hsa-miR-522 MIMAT0002868AAAAUGGUucccuuuagagugu 450 hsa-miR-519a* MIMAT0005452CUCUAGAGggaagcgcuuucug 414 hsa-miR-519a MIMAT0002869AAAGUGCAuccuuuuagagugu 451 hsa-miR-527 MIMAT0002862 CUGCAAAGggaagcccuuuc445 hsa-miR-516a-5p MIMAT0004770 UUCUCGAGgaaagaagcacuuuc 452hsa-miR-516a-3p MIMAT0006778 UGCUUCCUuucagagggu 443 hsa-miR-499-5pMIMAT0002870 UUAAGACUugcagugauguuu 453 hsa-miR-499-3p MIMAT0004772AACAUCACagcaagucugugcu 454 hsa-miR-500 MIMAT0004773UAAUCCUUgcuaccugggugaga 455 hsa-miR-500* MIMAT0002871AUGCACCUgggcaaggauucug 456 hsa-miR-501-5p MIMAT0002872AAUCCUUUgucccugggugaga 457 hsa-miR-501-3p MIMAT0004774AAUGCACCcgggcaaggauucu 458 hsa-miR-502-5p MIMAT0002873AUCCUUGCuaucugggugcua 459 hsa-miR-502-3p MIMAT0004775AAUGCACCugggcaaggauuca 460 hsa-miR-503 MIMAT0002874UAGCAGCGggaacaguucugcag 461 hsa-miR-504 MIMAT0002875AGACCCUGgucugcacucuauc 462 hsa-miR-505* MIMAT0004776GGGAGCCAggaaguauugaugu 463 hsa-miR-505 MIMAT0002876CGUCAACAcuugcugguuuccu 464 hsa-miR-513a-5p MIMAT0002877UUCACAGGgaggugucau 465 hsa-miR-513a-3p MIMAT0004777UAAAUUUCaccuuucugagaagg 466 hsa-miR-506 MIMAT0002878UAAGGCACccuucugaguaga 467 hsa-miR-507 MIMAT0002879 UUUUGCACcuuuuggagugaa468 hsa-miR-508-5p MIMAT0004778 UACUCCAGagggcgucacucaug 469hsa-miR-508-3p MIMAT0002880 UGAUUGUAgccuuuuggaguaga 470 hsa-miR-509-5pMIMAT0004779 UACUGCAGacaguggcaauca 471 hsa-miR-509-3p MIMAT0002881UGAUUGGUacgucuguggguag 472 hsa-miR-510 MIMAT0002882UACUCAGGagaguggcaaucac 473 hsa-miR-514 MIMAT0002883AUUGACACuucugugaguaga 474 hsa-miR-532-5p MIMAT0002888CAUGCCUUgaguguaggaccgu 475 hsa-miR-532-3p MIMAT0004780CCUCCCACacccaaggcuugca 476 hsa-miR-455-5p MIMAT0003150UAUGUGCCuuuggacuacaucg 477 hsa-miR-455-3p MIMAT0004784GCAGUCCAugggcauauacac 478 hsa-miR-539 MIMAT0003163GGAGAAAUuauccuuggugugu 479 hsa-miR-544 MIMAT0003164AUUCUGCAuuuuuagcaaguuc 480 hsa-miR-545* MIMAT0004785UCAGUAAAuguuuauuagauga 481 hsa-miR-545 MIMAT0003165UCAGCAAAcauuuauugugugc 482 hsa-miR-487b MIMAT0003180AAUCGUACagggucauccacuu 483 hsa-miR-551a MIMAT0003214GCGACCCAcucuugguuucca 484 hsa-miR-552 MIMAT0003215 AACAGGUGacugguuagacaa485 hsa-miR-553 MIMAT0003216 AAAACGGUgagauuuuguuuu 486 hsa-miR-554MIMAT0003217 GCUAGUCCugacucagccagu 487 hsa-miR-92b* MIMAT0004792AGGGACGGgacgcggugcagug 488 hsa-miR-92b MIMAT0003218UAUUGCACucgucccggccucc 489 hsa-miR-555 MIMAT0003219AGGGUAAGcugaaccucugau 490 hsa-miR-556-5p MIMAT0003220GAUGAGCUcauuguaauaugag 491 hsa-miR-556-3p MIMAT0004793AUAUUACCauuagcucaucuuu 492 hsa-miR-557 MIMAT0003221GUUUGCACgggugggccuugucu 493 hsa-miR-558 MIMAT0003222 UGAGCUGCuguaccaaaau494 hsa-miR-559 MIMAT0003223 UAAAGUAAauaugcaccaaaa 495 hsa-miR-561MIMAT0003225 CAAAGUUUaagauccuugaagu 496 hsa-miR-562 MIMAT0003226AAAGUAGCuguaccauuugc 497 hsa-miR-563 MIMAT0003227 AGGUUGACauacguuuccc498 hsa-miR-564 MIMAT0003228 AGGCACGGugucagcaggc 499 hsa-miR-566MIMAT0003230 GGGCGCCUgugaucccaac 500 hsa-miR-567 MIMAT0003231AGUAUGUUcuuccaggacagaac 501 hsa-miR-568 MIMAT0003232AUGUAUAAauguauacacac 502 hsa-miR-551b* MIMAT0004794GAAAUCAAgcgugggugagacc 503 hsa-miR-551b MIMAT0003233GCGACCCAuacuugguuucag 504 hsa-miR-569 MIMAT0003234 AGUUAAUGaauccuggaaagu505 hsa-miR-570 MIMAT0003235 CGAAAACAgcaauuaccuuugc 506 hsa-miR-571MIMAT0003236 UGAGUUGGccaucugagugag 507 hsa-miR-572 MIMAT0003237GUCCGCUCggcgguggccca 508 hsa-miR-573 MIMAT0003238CUGAAGUGauguguaacugaucag 509 hsa-miR-574-5p MIMAT0004795UGAGUGUGugugugugagugugu 510 hsa-miR-574-3p MIMAT0003239CACGCUCAugcacacacccaca 511 hsa-miR-575 MIMAT0003240 GAGCCAGUuggacaggagc512 hsa-miR-576-5p MIMAT0003241 AUUCUAAUuucuccacgucuuu 513hsa-miR-576-3p MIMAT0004796 AAGAUGUGgaaaaauuggaauc 514 hsa-miR-577MIMAT0003242 UAGAUAAAauauugguaccug 515 hsa-miR-578 MIMAT0003243CUUCUUGUgcucuaggauugu 516 hsa-miR-579 MIMAT0003244UUCAUUUGguauaaaccgcgauu 517 hsa-miR-580 MIMAT0003245UUGAGAAUgaugaaucauuagg 518 hsa-miR-581 MIMAT0003246UCUUGUGUucucuagaucagu 519 hsa-miR-582-5p MIMAT0003247UUACAGUUguucaaccaguuacu 520 hsa-miR-582-3p MIMAT0004797UAACUGGUugaacaacugaacc 521 hsa-miR-583 MIMAT0003248CAAAGAGGaaggucccauuac 522 hsa-miR-584 MIMAT0003249UUAUGGUUugccugggacugag 523 hsa-miR-585 MIMAT0003250 UGGGCGUAucuguaugcua524 hsa-miR-548a-3p MIMAT0003251 CAAAACUGgcaauuacuuuugc 525 hsa-miR-586MIMAT0003252 UAUGCAUUguauuuuuaggucc 526 hsa-miR-587 MIMAT0003253UUUCCAUAggugaugagucac 527 hsa-miR-548b-5p MIMAT0004798AAAAGUAAuugugguuuuggcc 528 hsa-miR-548b-3p MIMAT0003254CAAGAACCucaguugcuuuugu 529 hsa-miR-588 MIMAT0003255UUGGCCACaauggguuagaac 530 hsa-miR-589 MIMAT0004799UGAGAACCacgucugcucugag 531 hsa-miR-589* MIMAT0003256UCAGAACAaaugccgguucccaga 532 hsa-miR-550 MIMAT0004800AGUGCCUGagggaguaagagccc 533 hsa-miR-550* MIMAT0003257UGUCUUACucccucaggcacau 534 hsa-miR-590-5p MIMAT0003258GAGCUUAUucauaaaagugcag 535 hsa-miR-590-3p MIMAT0004801UAAUUUUAuguauaagcuagu 536 hsa-miR-591 MIMAT0003259 AGACCAUGgguucucauugu537 hsa-miR-592 MIMAT0003260 UUGUGUCAauaugcgaugaugu 538 hsa-miR-593*MIMAT0003261 AGGCACCAgccaggcauugcucagc 539 hsa-miR-593 MIMAT0004802UGUCUCUGcugggguuucu 540 hsa-miR-595 MIMAT0003263 GAAGUGUGccguggugugucu541 hsa-miR-596 MIMAT0003264 AAGCCUGCccggcuccucggg 542 hsa-miR-597MIMAT0003265 UGUGUCACucgaugaccacugu 543 hsa-miR-598 MIMAT0003266UACGUCAUcguugucaucguca 544 hsa-miR-599 MIMAT0003267 GUUGUGUCaguuuaucaaac545 hsa-miR-548a-5p MIMAT0004803 AAAAGUAAuugcgaguuuuacc 546 hsa-miR-600MIMAT0003268 ACUUACAGacaagagccuugcuc 547 hsa-miR-601 MIMAT0003269UGGUCUAGgauuguuggaggag 548 hsa-miR-602 MIMAT0003270GACACGGGcgacagcugcggccc 549 hsa-miR-603 MIMAT0003271CACACACUgcaauuacuuuugc 550 hsa-miR-604 MIMAT0003272 AGGCUGCGgaauucaggac551 hsa-miR-605 MIMAT0003273 UAAAUCCCauggugccuucuccu 552 hsa-miR-606MIMAT0003274 AAACUACUgaaaaucaaagau 553 hsa-miR-607 MIMAT0003275GUUCAAAUccagaucuauaac 554 hsa-miR-608 MIMAT0003276AGGGGUGGuguugggacagcuccgu 555 hsa-miR-609 MIMAT0003277AGGGUGUUucucucaucucu 556 hsa-miR-610 MIMAT0003278 UGAGCUAAaugugugcuggga557 hsa-miR-611 MIMAT0003279 GCGAGGACcccucggggucugac 558 hsa-miR-612MIMAT0003280 GCUGGGCAgggcuucugagcuccuu 559 hsa-miR-613 MIMAT0003281AGGAAUGUuccuucuuugcc 560 hsa-miR-614 MIMAT0003282GAACGCCUguucuugccaggugg 561 hsa-miR-615-5p MIMAT0004804GGGGGUCCccggugcucggauc 562 hsa-miR-615-3p MIMAT0003283UCCGAGCCugggucucccucuu 563 hsa-miR-616* MIMAT0003284ACUCAAAAcccuucagugacuu 564 hsa-miR-616 MIMAT0004805AGUCAUUGgaggguuugagcag 565 hsa-miR-548c-5p MIMAT0004806AAAAGUAAuugcgguuuuugcc 566 hsa-miR-548c-3p MIMAT0003285CAAAAAUCucaauuacuuuugc 567 hsa-miR-617 MIMAT0003286AGACUUCCcauuugaagguggc 568 hsa-miR-618 MIMAT0003287AAACUCUAcuuguccuucugagu 569 hsa-miR-619 MIMAT0003288GACCUGGAcauguuugugcccagu 570 hsa-miR-620 MIMAT0003289AUGGAGAUagauauagaaau 571 hsa-miR-621 MIMAT0003290 GGCUAGCAacagcgcuuaccu572 hsa-miR-622 MIMAT0003291 ACAGUCUGcugagguuggagc 573 hsa-miR-623MIMAT0003292 AUCCCUUGcaggggcuguugggu 574 hsa-miR-624* MIMAT0003293UAGUACCAguaccuuguguuca 575 hsa-miR-624 MIMAT0004807CACAAGGUauugguauuaccu 576 hsa-miR-625 MIMAT0003294 AGGGGGAAaguucuauagucc577 hsa-miR-625* MIMAT0004808 GACUAUAGaacuuucccccuca 578 hsa-miR-626MIMAT0003295 AGCUGUCUgaaaaugucuu 579 hsa-miR-627 MIMAT0003296GUGAGUCUcuaagaaaagagga 580 hsa-miR-628-5p MIMAT0004809AUGCUGACauauuuacuagagg 581 hsa-miR-628-3p MIMAT0003297UCUAGUAAgaguggcagucga 582 hsa-miR-629 MIMAT0004810 UGGGUUUAcguugggagaacu583 hsa-miR-629* MIMAT0003298 GUUCUCCCaacguaagcccagc 584 hsa-miR-630MIMAT0003299 AGUAUUCUguaccagggaaggu 585 hsa-miR-631 MIMAT0003300AGACCUGGcccagaccucagc 586 hsa-miR-33b MIMAT0003301 GUGCAUUGcuguugcauugc587 hsa-miR-33b* MIMAT0004811 CAGUGCCUcggcagugcagccc 588 hsa-miR-632MIMAT0003302 GUGUCUGCuuccuguggga 589 hsa-miR-633 MIMAT0003303CUAAUAGUaucuaccacaauaaa 590 hsa-miR-634 MIMAT0003304AACCAGCAccccaacuuuggac 591 hsa-miR-635 MIMAT0003305ACUUGGGCacugaaacaaugucc 592 hsa-miR-636 MIMAT0003306UGUGCUUGcucgucccgcccgca 593 hsa-miR-637 MIMAT0003307ACUGGGGGcuuucgggcucugcgu 594 hsa-miR-638 MIMAT0003308AGGGAUCGcgggcggguggcggccu 595 hsa-miR-639 MIMAT0003309AUCGCUGCgguugcgagcgcugu 596 hsa-miR-640 MIMAT0003310AUGAUCCAggaaccugccucu 597 hsa-miR-641 MIMAT0003311AAAGACAUaggauagagucaccuc 598 hsa-miR-642 MIMAT0003312GUCCCUCUccaaaugugucuug 599 hsa-miR-643 MIMAT0003313ACUUGUAUgcuagcucagguag 600 hsa-miR-644 MIMAT0003314 AGUGUGGCuuucuuagagc601 hsa-miR-645 MIMAT0003315 UCUAGGCUgguacugcuga 602 hsa-miR-646MIMAT0003316 AAGCAGCUgccucugaggc 603 hsa-miR-647 MIMAT0003317GUGGCUGCacucacuuccuuc 604 hsa-miR-648 MIMAT0003318 AAGUGUGCagggcacuggu605 hsa-miR-649 MIMAT0003319 AAACCUGUguuguucaagaguc 606 hsa-miR-650MIMAT0003320 AGGAGGCAgcgcucucaggac 607 hsa-miR-651 MIMAT0003321UUUAGGAUaagcuugacuuuug 608 hsa-miR-652 MIMAT0003322AAUGGCGCcacuaggguugug 609 hsa-miR-548d-5p MIMAT0004812AAAAGUAAuugugguuuuugcc 610 hsa-miR-548d-3p MIMAT0003323CAAAAACCacaguuucuuuugc 611 hsa-miR-661 MIMAT0003324UGCCUGGGucucuggccugcgcgu 612 hsa-miR-662 MIMAT0003325UCCCACGUuguggcccagcag 613 hsa-miR-663 MIMAT0003326AGGCGGGGcgccgcgggaccgc 614 hsa-miR-449b MIMAT0003327AGGCAGUGuauuguuagcuggc 615 hsa-miR-449b* MIMAT0009203CAGCCACAacuacccugccacu 616 hsa-miR-653 MIMAT0003328GUGUUGAAacaaucucuacug 617 hsa-miR-411 MIMAT0003329 UAGUAGACcguauagcguacg618 hsa-miR-411* MIMAT0004813 UAUGUAACacgguccacuaacc 619 hsa-miR-654-5pMIMAT0003330 UGGUGGGCcgcagaacaugugc 620 hsa-miR-654-3p MIMAT0004814UAUGUCUGcugaccaucaccuu 621 hsa-miR-655 MIMAT0003331AUAAUACAugguuaaccucuuu 622 hsa-miR-656 MIMAT0003332AAUAUUAUacagucaaccucu 623 hsa-miR-549 MIMAT0003333 UGACAACUauggaugagcucu624 hsa-miR-657 MIMAT0003335 GGCAGGUUcucacccucucuagg 625 hsa-miR-658MIMAT0003336 GGCGGAGGgaaguagguccguuggu 626 hsa-miR-659 MIMAT0003337CUUGGUUCagggagggucccca 627 hsa-miR-660 MIMAT0003338UACCCAUUgcauaucggaguug 628 hsa-miR-421 MIMAT0003339AUCAACAGacauuaauugggcgc 629 hsa-miR-542-5p MIMAT0003340UCGGGGAUcaucaugucacgaga 630 hsa-miR-542-3p MIMAT0003389UGUGACAGauugauaacugaaa 631 hsa-miR-758 MIMAT0003879UUUGUGACcugguccacuaacc 632 hsa-miR-1264 MIMAT0005791CAAGUCUUauuugagcaccuguu 633 hsa-miR-671-5p MIMAT0003880AGGAAGCCcuggaggggcuggag 634 hsa-miR-671-3p MIMAT0004819UCCGGUUCucagggcuccacc 635 hsa-miR-668 MIMAT0003881UGUCACUCggcucggcccacuac 636 hsa-miR-767-5p MIMAT0003882UGCACCAUgguugucugagcaug 637 hsa-miR-767-3p MIMAT0003883UCUGCUCAuaccccaugguuucu 638 hsa-miR-1224-5p MIMAT0005458GUGAGGACucgggaggugg 639 hsa-miR-1224-3p MIMAT0005459CCCCACCUccucucuccucag 640 hsa-miR-320b MIMAT0005792AAAAGCUGgguugagagggcaa 641 hsa-miR-320c MIMAT0005793AAAAGCUGgguugagagggu 642 hsa-miR-1296 MIMAT0005794UUAGGGCCcuggcuccaucucc 643 hsa-miR-1468 MIMAT0006789CUCCGUUUgccuguuucgcug 644 hsa-miR-1323 MIMAT0005795UCAAAACUgaggggcauuuucu 645 hsa-miR-1271 MIMAT0005796CUUGGCACcuagcaagcacuca 646 hsa-miR-1301 MIMAT0005797UUGCAGCUgccugggagugacuuc 647 hsa-miR-454* MIMAT0003884ACCCUAUCaauauugucucugc 648 hsa-miR-454 MIMAT0003885UAGUGCAAuauugcuuauagggu 649 hsa-miR-1185 MIMAT0005798AGAGGAUAcccuuuguauguu 650 hsa-miR-449c MIMAT0010251UAGGCAGUguauugcuagcggcugu 651 hsa-miR-449c* MIMAT0013771UUGCUAGUugcacuccucucugu 652 hsa-miR-1283 MIMAT0005799UCUACAAAggaaagcgcuuucu 653 hsa-miR-769-5p MIMAT0003886UGAGACCUcuggguucugagcu 654 hsa-miR-769-3p MIMAT0003887CUGGGAUCuccggggucuugguu 655 hsa-miR-766 MIMAT0003888ACUCCAGCcccacagccucagc 656 hsa-miR-762 MIMAT0010313GGGGCUGGggccggggccgagc 657 hsa-miR-802 MIMAT0004185CAGUAACAaagauucauccuugu 658 hsa-miR-670 MIMAT0010357GUCCCUGAguguauguggug 659 hsa-miR-1298 MIMAT0005800UUCAUUCGgcuguccagaugua 660 hsa-miR-2113 MIMAT0009206AUUUGUGCuuggcucugucac 661 hsa-miR-761 MIMAT0010364GCAGCAGGgugaaacugacaca 662 hsa-miR-764 MIMAT0010367GCAGGUGCucacuuguccuccu 663 hsa-miR-759 MIMAT0010497GCAGAGUGcaaacaauuuugac 664 hsa-miR-765 MIMAT0003945UGGAGGAGaaggaaggugaug 665 hsa-miR-770-5p MIMAT0003948UCCAGUACcacgugucagggcca 666 hsa-miR-675 MIMAT0004284UGGUGCGGagagggcccacagug 667 hsa-miR-675* MIMAT0006790CUGUAUGCccucaccgcuca 668 hsa-miR-298 MIMAT0004901AGCAGAAGcagggagguucuccca 669 hsa-miR-891a MIMAT0004902UGCAACGAaccugagccacuga 670 hsa-miR-300 MIMAT0004903UAUACAAGggcagacucucucu 671 hsa-miR-886-5p MIMAT0004905CGGGUCGGaguuagcucaagcgg 672 hsa-miR-886-3p MIMAT0004906CGCGGGUGcuuacugacccuu 673 hsa-miR-892a MIMAT0004907CACUGUGUccuuucugcguag 674 hsa-miR-220b MIMAT0004908CCACCACCgugucugacacuu 675 hsa-miR-450b-5p MIMAT0004909UUUUGCAAuauguuccugaaua 676 hsa-miR-450b-3p MIMAT0004910UUGGGAUCauuuugcauccaua 677 hsa-miR-874 MIMAT0004911CUGCCCUGgcccgagggaccga 678 hsa-miR-890 MIMAT0004912UACUUGGAaaggcaucaguug 679 hsa-miR-891b MIMAT0004913UGCAACUUaccugagucauuga 680 hsa-miR-220c MIMAT0004915ACACAGGGcuguugugaagacu 681 hsa-miR-888 MIMAT0004916UACUCAAAaagcugucaguca 682 hsa-miR-888* MIMAT0004917GACUGACAccucuuugggugaa 683 hsa-miR-892b MIMAT0004918CACUGGCUccuuucuggguaga 684 hsa-miR-541* MIMAT0004919AAAGGAUUcugcugucggucccacu 685 hsa-miR-541 MIMAT0004920UGGUGGGCacagaaucuggacu 686 hsa-miR-889 MIMAT0004921UUAAUAUCggacaaccauugu 687 hsa-miR-875-5p MIMAT0004922UAUACCUCaguuuuaucaggug 688 hsa-miR-875-3p MIMAT0004923CCUGGAAAcacugagguugug 689 hsa-miR-876-5p MIMAT0004924UGGAUUUCuuugugaaucacca 690 hsa-miR-876-3p MIMAT0004925UGGUGGUUuacaaaguaauuca 691 hsa-miR-708 MIMAT0004926AAGGAGCUuacaaucuagcuggg 692 hsa-miR-708* MIMAT0004927CAACUAGAcugugagcuucuag 693 hsa-miR-147b MIMAT0004928GUGUGCGGaaaugcuucugcua 694 hsa-miR-190b MIMAT0004929UGAUAUGUuugauauuggguu 695 hsa-miR-744 MIMAT0004945UGCGGGGCuagggcuaacagca 696 hsa-miR-744* MIMAT0004946CUGUUGCCacuaaccucaaccu 697 hsa-miR-885-5p MIMAT0004947UCCAUUACacuacccugccucu 698 hsa-miR-885-3p MIMAT0004948AGGCAGCGggguguaguggaua 699 hsa-miR-877 MIMAT0004949 GUAGAGGAgauggcgcaggg700 hsa-miR-877* MIMAT0004950 UCCUCUUCucccuccucccag 701 hsa-miR-887MIMAT0004951 GUGAACGGgcgccaucccgagg 702 hsa-miR-665 MIMAT0004952ACCAGGAGgcugaggccccu 703 hsa-miR-873 MIMAT0004953 GCAGGAACuugugagucuccu704 hsa-miR-543 MIMAT0004954 AAACAUUCgcggugcacuucuu 705 hsa-miR-374bMIMAT0004955 AUAUAAUAcaaccugcuaagug 706 hsa-miR-374b* MIMAT0004956CUUAGCAGguuguauuaucauu 707 hsa-miR-760 MIMAT0004957 CGGCUCUGggucugugggga708 hsa-miR-301b MIMAT0004958 CAGUGCAAugauauugucaaagc 709 hsa-miR-216bMIMAT0004959 AAAUCUCUgcaggcaaauguga 710 hsa-miR-208b MIMAT0004960AUAAGACGaacaaaagguuugu 711 hsa-miR-920 MIMAT0004970 GGGGAGCUguggaagcagua712 hsa-miR-921 MIMAT0004971 CUAGUGAGggacagaaccaggauuc 713 hsa-miR-922MIMAT0004972 GCAGCAGAgaauaggacuacguc 714 hsa-miR-924 MIMAT0004974AGAGUCUUgugaugucuugc 715 hsa-miR-509-3-5p MIMAT0004975UACUGCAGacguggcaaucaug 716 hsa-miR-933 MIMAT0004976UGUGCGCAgggagaccucuccc 717 hsa-miR-934 MIMAT0004977UGUCUACUacuggagacacugg 718 hsa-miR-935 MIMAT0004978CCAGUUACcgcuuccgcuaccgc 719 hsa-miR-936 MIMAT0004979ACAGUAGAgggaggaaucgcag 720 hsa-miR-937 MIMAT0004980AUCCGCGCucugacucucugcc 721 hsa-miR-938 MIMAT0004981UGCCCUUAaaggugaacccagu 722 hsa-miR-939 MIMAT0004982UGGGGAGCugaggcucugggggug 723 hsa-miR-940 MIMAT0004983AAGGCAGGgcccccgcucccc 724 hsa-miR-941 MIMAT0004984CACCCGGCugugugcacaugugc 725 hsa-miR-942 MIMAT0004985UCUUCUCUguuuuggccaugug 726 hsa-miR-943 MIMAT0004986CUGACUGUugccguccuccag 727 hsa-miR-944 MIMAT0004987AAAUUAUUguacaucggaugag 728 hsa-miR-297 MIMAT0004450AUGUAUGUgugcaugugcaug 729 hsa-miR-1178 MIMAT0005823UUGCUCACuguucuucccuag 730 hsa-miR-1179 MIMAT0005824AAGCAUUCuuucauugguugg 731 hsa-miR-1180 MIMAT0005825UUUCCGGCucgcgugggugugu 732 hsa-miR-1181 MIMAT0005826CCGUCGCCgccacccgagccg 733 hsa-miR-1182 MIMAT0005827GAGGGUCUugggagggaugugac 734 hsa-miR-1183 MIMAT0005828CACUGUAGgugauggugagagugggca 735 hsa-miR-1184 MIMAT0005829CCUGCAGCgacuugauggcuucc 736 hsa-miR-1225-5p MIMAT0005572GUGGGUACggcccagugggggg 737 hsa-miR-1225-3p MIMAT0005573UGAGCCCCugugccgcccccag 738 hsa-miR-1226* MIMAT0005576GUGAGGGCaugcaggccuggaugggg 739 hsa-miR-1226 MIMAT0005577UCACCAGCccuguguucccuag 740 hsa-miR-1227 MIMAT0005580CGUGCCACccuuuuccccag 741 hsa-miR-1228* MIMAT0005582GUGGGCGGgggcaggugugug 742 hsa-miR-1228 MIMAT0005583 UCACACCUgccucgcccccc743 hsa-miR-1229 MIMAT0005584 CUCUCACCacugcccucccacag 744 hsa-miR-1231MIMAT0005586 GUGUCUGGgcggacagcugc 745 hsa-miR-1233 MIMAT0005588UGAGCCCUguccucccgcag 746 hsa-miR-1234 MIMAT0005589UCGGCCUGaccacccaccccac 747 hsa-miR-1236 MIMAT0005591CCUCUUCCccuugucucuccag 748 hsa-miR-1237 MIMAT0005592UCCUUCUGcuccgucccccag 749 hsa-miR-1238 MIMAT0005593 CUUCCUCGucugucugcccc750 hsa-miR-1200 MIMAT0005863 CUCCUGAGccauucugagccuc 751 hsa-miR-1201MIMAT0005864 AGCCUGAUuaaacacaugcucuga 752 hsa-miR-1202 MIMAT0005865GUGCCAGCugcagugggggag 753 hsa-miR-1203 MIMAT0005866CCCGGAGCcaggaugcagcuc 754 hsa-miR-663b MIMAT0005867GGUGGCCCggccgugccugagg 755 hsa-miR-1204 MIMAT0005868UCGUGGCCuggucuccauuau 756 hsa-miR-1205 MIMAT0005869 UCUGCAGGguuugcuuugag757 hsa-miR-1206 MIMAT0005870 UGUUCAUGuagauguuuaagc 758 hsa-miR-1207-5pMIMAT0005871 UGGCAGGGaggcugggagggg 759 hsa-miR-1207-3p MIMAT0005872UCAGCUGGcccucauuuc 760 hsa-miR-1208 MIMAT0005873 UCACUGUUcagacaggcgga761 hsa-miR-548e MIMAT0005874 AAAAACUGagacuacuuuugca 762 hsa-miR-548jMIMAT0005875 AAAAGUAAuugcggucuuuggu 763 hsa-miR-1285 MIMAT0005876UCUGGGCAacaaagugagaccu 764 hsa-miR-1286 MIMAT0005877UGCAGGACcaagaugagcccu 765 hsa-miR-1287 MIMAT0005878UGCUGGAUcagugguucgaguc 766 hsa-miR-1289 MIMAT0005879UGGAGUCCaggaaucugcauuuu 767 hsa-miR-1290 MIMAT0005880UGGAUUUUuggaucaggga 768 hsa-miR-1291 MIMAT0005881UGGCCCUGacugaagaccagcagu 769 hsa-miR-548k MIMAT0005882AAAAGUACuugcggauuuugcu 770 hsa-miR-1293 MIMAT0005883UGGGUGGUcuggagauuugugc 771 hsa-miR-1294 MIMAT0005884UGUGAGGUuggcauuguugucu 772 hsa-miR-1295 MIMAT0005885UUAGGCCGcagaucuggguga 773 hsa-miR-1297 MIMAT0005886 UUCAAGUAauucaggug774 hsa-miR-1299 MIMAT0005887 UUCUGGAAuucugugugaggga 775 hsa-miR-5481MIMAT0005889 AAAAGUAUuugcggguuuuguc 776 hsa-miR-1302 MIMAT0005890UUGGGACAuacuuaugcuaaa 777 hsa-miR-1303 MIMAT0005891UUUAGAGAcggggucuugcucu 778 hsa-miR-1304 MIMAT0005892UUUGAGGCuacagugagaugug 779 hsa-miR-1305 MIMAT0005893UUUUCAACucuaaugggagaga 780 hsa-miR-1243 MIMAT0005894AACUGGAUcaauuauaggagug 781 hsa-miR-548f MIMAT0005895 AAAAACUGuaauuacuuuu782 hsa-miR-1244 MIMAT0005896 AAGUAGUUgguuuguaugagaugguu 783hsa-miR-1245 MIMAT0005897 AAGUGAUCuaaaggccuacau 784 hsa-miR-1246MIMAT0005898 AAUGGAUUuuuggagcagg 785 hsa-miR-1247 MIMAT0005899ACCCGUCCcguucguccccgga 786 hsa-miR-1248 MIMAT0005900ACCUUCUUguauaagcacugugcuaaa 787 hsa-miR-1249 MIMAT0005901ACGCCCUUcccccccuucuuca 788 hsa-miR-1250 MIMAT0005902ACGGUGCUggauguggccuuu 789 hsa-miR-1251 MIMAT0005903ACUCUAGCugccaaaggcgcu 790 hsa-miR-1253 MIMAT0005904AGAGAAGAagaucagccugca 791 hsa-miR-1254 MIMAT0005905AGCCUGGAagcuggagccugcagu 792 hsa-miR-1255a MIMAT0005906AGGAUGAGcaaagaaaguagauu 793 hsa-miR-1256 MIMAT0005907AGGCAUUGacuucucacuagcu 794 hsa-miR-1257 MIMAT0005908AGUGAAUGauggguucugacc 795 hsa-miR-1258 MIMAT0005909AGUUAGGAuuaggucguggaa 796 hsa-miR-1259 MIMAT0005910AUAUAUGAugacuuagcuuuu 797 hsa-miR-1260 MIMAT0005911 AUCCCACCucugccacca798 hsa-miR-548g MIMAT0005912 AAAACUGUaauuacuuuuguac 799 hsa-miR-1261MIMAT0005913 AUGGAUAAggcuuuggcuu 800 hsa-miR-1262 MIMAT0005914AUGGGUGAauuuguagaaggau 801 hsa-miR-1263 MIMAT0005915AUGGUACCcuggcauacugagu 802 hsa-miR-548n MIMAT0005916CAAAAGUAauuguggauuuugu 803 hsa-miR-548m MIMAT0005917CAAAGGUAuuugugguuuuug 804 hsa-miR-1265 MIMAT0005918CAGGAUGUggucaaguguuguu 805 hsa-miR-548o MIMAT0005919CCAAAACUgcaguuacuuuugc 806 hsa-miR-1266 MIMAT0005920CCUCAGGGcuguagaacagggcu 807 hsa-miR-1267 MIMAT0005921CCUGUUGAaguguaaucccca 808 hsa-miR-1268 MIMAT0005922 CGGGCGUGgugguggggg809 hsa-miR-1269 MIMAT0005923 CUGGACUGagccgugcuacugg 810 hsa-miR-1270MIMAT0005924 CUGGAGAUauggaagagcugugu 811 hsa-miR-1272 MIMAT0005925GAUGAUGAuggcagcaaauucugaaa 812 hsa-miR-1273 MIMAT0005926GGGCGACAaagcaagacucuuucuu 813 hsa-miR-1274a MIMAT0005927GUCCCUGUucaggcgcca 814 hsa-miR-548h MIMAT0005928 AAAAGUAAucgcgguuuuuguc815 hsa-miR-1275 MIMAT0005929 GUGGGGGAgaggcuguc 816 hsa-miR-1276MIMAT0005930 UAAAGAGCccuguggagaca 817 hsa-miR-302e MIMAT0005931UAAGUGCUuccaugcuu 818 hsa-miR-302f MIMAT0005932 UAAUUGCUuccauguuu 819hsa-miR-1277 MIMAT0005933 UACGUAGAuauauauguauuuu 820 hsa-miR-548pMIMAT0005934 UAGCAAAAacugcaguuacuuu 821 hsa-miR-548i MIMAT0005935AAAAGUAAuugcggauuuugcc 822 hsa-miR-1278 MIMAT0005936UAGUACUGugcauaucaucuau 823 hsa-miR-1279 MIMAT0005937 UCAUAUUGcuucuuucu824 hsa-miR-1274b MIMAT0005938 UCCCUGUUcgggcgcca 825 hsa-miR-1281MIMAT0005939 UCGCCUCCuccucuccc 826 hsa-miR-1282 MIMAT0005940UCGUUUGCcuuuuucugcuu 827 hsa-miR-1284 MIMAT0005941UCUAUACAgacccuggcuuuuc 828 hsa-miR-1288 MIMAT0005942UGGACUGCccugaucuggaga 829 hsa-miR-1292 MIMAT0005943UGGGAACGgguuccggcagacgcug 830 hsa-miR-1252 MIMAT0005944AGAAGGAAauugaauucauuua 831 hsa-miR-1255b MIMAT0005945CGGAUGAGcaaagaaagugguu 832 hsa-miR-1280 MIMAT0005946 UCCCACCGcugccaccc833 hsa-miR-1308 MIMAT0005947 GCAUGGGUgguucagugg 834 hsa-miR-664*MIMAT0005948 ACUGGCUAgggaaaaugauuggau 835 hsa-miR-664 MIMAT0005949UAUUCAUUuauccccagccuaca 836 hsa-miR-1306 MIMAT0005950 ACGUUGGCucugguggug837 hsa-miR-1307 MIMAT0005951 ACUCGGCGuggcgucggucgug 838 hsa-miR-513bMIMAT0005788 UUCACAAGgaggugucauuuau 839 hsa-miR-513c MIMAT0005789UUCUCAAGgaggugucguuuau 840 hsa-miR-1321 MIMAT0005952 CAGGGAGGugaaugugau841 hsa-miR-1322 MIMAT0005953 GAUGAUGCugcugaugcug 842 hsa-miR-720MIMAT0005954 UCUCGCUGgggccucca 843 hsa-miR-1197 MIMAT0005955UAGGACACauggucuacuucu 844 hsa-miR-1324 MIMAT0005956CCAGACAGaauucuaugcacuuuc 845 hsa-miR-1469 MIMAT0007347CUCGGCGCggggcgcgggcucc 846 hsa-miR-1470 MIMAT0007348GCCCUCCGcccgugcaccccg 847 hsa-miR-1471 MIMAT0007349GCCCGCGUguggagccaggugu 848 hsa-miR-1537 MIMAT0007399AAAACCGUcuaguuacaguugu 849 hsa-miR-1538 MIMAT0007400CGGCCCGGgcugcugcuguuccu 850 hsa-miR-1539 MIMAT0007401UCCUGCGCgucccagaugccc 851 hsa-miR-103-as MIMAT0007402UCAUAGCCcuguacaaugcugcu 852 hsa-miR-320d MIMAT0006764AAAAGCUGgguugagagga 853 hsa-miR-1825 MIMAT0006765 UCCAGUGCccuccucucc 854hsa-miR-1826 MIMAT0006766 AUUGAUCAucgacacuucgaacgcaau 855 hsa-miR-1827MIMAT0006767 UGAGGCAGuagauugaau 856 hsa-miR-1908 MIMAT0007881CGGCGGGGacggcgauugguc 857 hsa-miR-1909* MIMAT0007882UGAGUGCCggugccugcccug 858 hsa-miR-1909 MIMAT0007883CGCAGGGGccgggugcucaccg 859 hsa-miR-1910 MIMAT0007884CCAGUCCUgugccugccgccu 860 hsa-miR-1911 MIMAT0007885UGAGUACCgccaugucuguuggg 861 hsa-miR-1911* MIMAT0007886CACCAGGCauuguggucucc 862 hsa-miR-1912 MIMAT0007887UACCCAGAgcaugcagugugaa 863 hsa-miR-1913 MIMAT0007888UCUGCCCCcuccgcugcugcca 864 hsa-miR-1914 MIMAT0007889CCCUGUGCccggcccacuucug 865 hsa-miR-1914* MIMAT0007890GGAGGGGUcccgcacugggagg 866 hsa-miR-1915* MIMAT0007891ACCUUGCCuugcugcccgggcc 867 hsa-miR-1915 MIMAT0007892CCCCAGGGcgacgcggcggg 868 hsa-miR-1972 MIMAT0009447UCAGGCCAggcacaguggcuca 869 hsa-miR-1973 MIMAT0009448 ACCGUGCAaagguagcaua870 hsa-miR-1975 MIMAT0009450 CCCCCACAaccgcgcuugacuagcu 871 hsa-miR-1976MIMAT0009451 CCUCCUGCccuccuugcugu 872 hsa-miR-1979 MIMAT0009454CUCCCACUgcuucacuugacua 873 hsa-miR-2052 MIMAT0009977UGUUUUGAuaacaguaaugu 874 hsa-miR-2053 MIMAT0009978GUGUUAAUuaaaccucuauuuac 875 hsa-miR-2054 MIMAT0009979CUGUAAUAuaaauuuaauuuauu 876 hsa-miR-2110 MIMAT0010133UUGGGGAAacggccgcugagug 877 hsa-miR-2114 MIMAT0011156UAGUCCCUuccuugaagcgguc 878 hsa-miR-2114* MIMAT0011157CGAGCCUCaagcaagggacuu 879 hsa-miR-2115 MIMAT0011158AGCUUCCAugacuccugaugga 880 hsa-miR-2115* MIMAT0011159CAUCAGAAuucauggaggcuag 881 hsa-miR-2116 MIMAT0011160GGUUCUUAgcauaggaggucu 882 hsa-miR-2116* MIMAT0011161CCUCCCAUgccaagaacuccc 883 hsa-miR-2117 MIMAT0011162UGUUCUCUuugccaaggacag 884 hsa-miR-548q MIMAT0011163GCUGGUGCaaaaguaauggcgg 885 hsa-miR-2276 MIMAT0011775UCUGCAAGugucagaggcgagg 886 hsa-miR-2277 MIMAT0011777UGACAGCGcccugccuggcuc 887 hsa-miR-2278 MIMAT0011778GAGAGCAGuguguguugccugg 888 hsa-miR-711 MIMAT0012734GGGACCCAgggagagacguaag 889 hsa-miR-718 MIMAT0012735CUUCCGCCccgccgggcgucg 890 hsa-miR-2861 MIMAT0013802 GGGGCCUGgcggugggcgg891 hsa-miR-2909 MIMAT0013863 GUUAGGGCcaacaucucuugg 892 hsa-miR-3115MIMAT0014977 AUAUGGGUuuacuaguuggu 893 hsa-miR-3116 MIMAT0014978UGCCUGGAacauaguagggacu 894 hsa-miR-3117 MIMAT0014979AUAGGACUcauauagugccag 895 hsa-miR-3118 MIMAT0014980UGUGACUGcauuaugaaaauucu 896 hsa-miR-3119 MIMAT0014981UGGCUUUUaacuuugauggc 897 hsa-miR-3120 MIMAT0014982 CACAGCAAguguagacaggca898 hsa-miR-3121 MIMAT0014983 UAAAUAGAguaggcaaaggaca 899 hsa-miR-3122MIMAT0014984 GUUGGGACaagaggacggucuu 900 hsa-miR-3123 MIMAT0014985CAGAGAAUuguuuaauc 901 hsa-miR-3124 MIMAT0014986 UUCGCGGGcgaaggcaaaguc902 hsa-miR-548s MIMAT0014987 AUGGCCAAaacugcaguuauuuu 903 hsa-miR-3125MIMAT0014988 UAGAGGAAgcuguggagaga 904 hsa-miR-3126-5p MIMAT0014989UGAGGGACagaugccagaagca 905 hsa-miR-3126-3p MIMAT0015377CAUCUGGCauccgucacacaga 906 hsa-miR-3127 MIMAT0014990AUCAGGGCuuguggaaugggaag 907 hsa-miR-3128 MIMAT0014991UCUGGCAAguaaaaaacucucau 908 hsa-miR-3129 MIMAT0014992GCAGUAGUguagagauugguuu 909 hsa-miR-3130-5p MIMAT0014995UACCCAGUcuccggugcagcc 910 hsa-miR-3130-3p MIMAT0014994GCUGCACCggagacuggguaa 911 hsa-miR-3131 MIMAT0014996UCGAGGACugguggaagggccuu 912 hsa-miR-3132 MIMAT0014997UGGGUAGAgaaggagcucagagga 913 hsa-miR-3133 MIMAT0014998UAAAGAACucuuaaaacccaau 914 hsa-miR-378b MIMAT0014999 ACUGGACUuggaggcagaa915 hsa-miR-3134 MIMAT0015000 UGAUGGAUaaaagacuacauauu 916 hsa-miR-3135MIMAT0015001 UGCCUAGGcugagacugcagug 917 hsa-miR-466 MIMAT0015002AUACACAUacacgcaacacacau 918 hsa-miR-3136 MIMAT0015003CUGACUGAauagguagggucauu 919 hsa-miR-544b MIMAT0015004ACCUGAGGuugugcauuucuaa 920 hsa-miR-3137 MIMAT0015005UCUGUAGCcugggagcaauggggu 921 hsa-miR-3138 MIMAT0015006UGUGGACAgugagguagagggagu 922 hsa-miR-3139 MIMAT0015007UAGGAGCUcaacagaugccuguu 923 hsa-miR-3140 MIMAT0015008AGCUUUUGggaauucagguagu 924 hsa-miR-548t MIMAT0015009CAAAAGUGaucgugguuuuug 925 hsa-miR-3141 MIMAT0015010 GAGGGCGGguggaggagga926 hsa-miR-3142 MIMAT0015011 AAGGCCUUucugaaccuucaga 927 hsa-miR-3143MIMAT0015012 AUAACAUUguaaagcgcuucuuucg 928 hsa-miR-548u MIMAT0015013CAAAGACUgcaauuacuuuugcg 929 hsa-miR-3144-5p MIMAT0015014AGGGGACCaaagagauauauag 930 hsa-miR-3144-3p MIMAT0015015AUAUACCUguucggucucuuua 931 hsa-miR-3145 MIMAT0015016AGAUAUUUugaguguuuggaauug 932 hsa-miR-1273c MIMAT0015017GGCGACAAaacgagacccuguc 933 hsa-miR-3146 MIMAT0015018CAUGCUAGgauagaaagaaugg 934 hsa-miR-3147 MIMAT0015019GGUUGGGCagugaggaggguguga 935 hsa-miR-548v MIMAT0015020AGCUACAGuuacuuuugcacca 936 hsa-miR-3148 MIMAT0015021UGGAAAAAacuggugugugcuu 937 hsa-miR-3149 MIMAT0015022UUUGUAUGgauauguguguguau 938 hsa-miR-3150 MIMAT0015023CUGGGGAGauccucgagguugg 939 hsa-miR-3151 MIMAT0015024GGUGGGGCaaugggaucaggu 940 hsa-miR-3152 MIMAT0015025UGUGUUAGaauaggggcaauaa 941 hsa-miR-3153 MIMAT0015026GGGGAAAGcgaguagggacauuu 942 hsa-miR-3074 MIMAT0015027GAUAUCAGcucaguaggcaccg 943 hsa-miR-3154 MIMAT0015028CAGAAGGGgaguugggagcaga 944 hsa-miR-3155 MIMAT0015029CCAGGCUCugcagugggaacu 945 hsa-miR-3156 MIMAT0015030AAAGAUCUggaagugggagaca 946 hsa-miR-3157 MIMAT0015031UUCAGCCAggcuagugcagucu 947 hsa-miR-3158 MIMAT0015032AAGGGCUUccucucugcaggac 948 hsa-miR-3159 MIMAT0015033UAGGAUUAcaagugucggccac 949 hsa-miR-3160 MIMAT0015034AGAGCUGAgacuagaaagccca 950 hsa-miR-3161 MIMAT0015035CUGAUAAGaacagaggcccagau 951 hsa-miR-3162 MIMAT0015036UUAGGGAGuagaaggguggggag 952 hsa-miR-3163 MIMAT0015037UAUAAAAUgagggcaguaagac 953 hsa-miR-3164 MIMAT0015038UGUGACUUuaagggaaauggcg 954 hsa-miR-3165 MIMAT0015039AGGUGGAUgcaaugugaccuca 955 hsa-miR-3166 MIMAT0015040CGCAGACAaugccuacuggccua 956 hsa-miR-1260b MIMAT0015041AUCCCACCacugccaccau 957 hsa-miR-3167 MIMAT0015042 AGGAUUUCagaaauacuggugu958 hsa-miR-3168 MIMAT0015043 GAGUUCUAcagucagac 959 hsa-miR-3169MIMAT0015044 UAGGACUGugcuuggcacauag 960 hsa-miR-3170 MIMAT0015045CUGGGGUUcugagacagacagu 961 hsa-miR-3171 MIMAT0015046AGAUGUAUggaaucuguauauauc 962 hsa-miR-3172 MIMAT0015047UGGGGUUUugcaguccuua 963 hsa-miR-3173 MIMAT0015048 AAAGGAGGaaauaggcaggcca964 hsa-miR-1193 MIMAT0015049 GGGAUGGUagaccggugacgugc 965hsa-miR-323b-5p MIMAT0001630 AGGUUGUCcguggugaguucgca 966 hsa-miR-323b-3pMIMAT0015050 CCCAAUACacggucgaccucuu 967 hsa-miR-3174 MIMAT0015051UAGUGAGUuagagaugcagagcc 968 hsa-miR-3175 MIMAT0015052CGGGGAGAgaacgcagugacgu 969 hsa-miR-3176 MIMAT0015053 ACUGGCCUgggacuaccgg970 hsa-miR-3177 MIMAT0015054 UGCACGGCacuggggacacgu 971 hsa-miR-3178MIMAT0015055 GGGGCGCGgccggaucg 972 hsa-miR-3179 MIMAT0015056AGAAGGGGugaaauuuaaacgu 973 hsa-miR-3180-5p MIMAT0015057CUUCCAGAcgcuccgccccacgucg 974 hsa-miR-3180-3p MIMAT0015058UGGGGCGGagcuuccggaggcc 975 hsa-miR-548w MIMAT0015060AAAAGUAAcugcgguuuuugccu 976 hsa-miR-3181 MIMAT0015061AUCGGGCCcucggcgccgg 977 hsa-miR-3182 MIMAT0015062 GCUUCUGUaguguaguc 978hsa-miR-3183 MIMAT0015063 GCCUCUCUcggagucgcucgga 979 hsa-miR-3184MIMAT0015064 UGAGGGGCcucagaccgagcuuuu 980 hsa-miR-3185 MIMAT0015065AGAAGAAGgcggucggucugcgg 981 hsa-miR-3065-5p MIMAT0015066UCAACAAAaucacugaugcugga 982 hsa-miR-3065-3p MIMAT0015378UCAGCACCaggauauuguuggag 983 hsa-miR-3186-5p MIMAT0015067CAGGCGUCugucuacguggcuu 984 hsa-miR-3186-3p MIMAT0015068UCACGCGGagagauggcuuug 985 hsa-miR-3187 MIMAT0015069 UUGGCCAUggggcugcgcgg986 hsa-miR-3188 MIMAT0015070 AGAGGCUUugugcggauacgggg 987 hsa-miR-3189MIMAT0015071 CCCUUGGGucugaugggguag 988 hsa-miR-320e MIMAT0015072AAAGCUGGguugagaagg 989 hsa-miR-3190-5p MIMAT0015073UGUGGAAGguagacggccagaga 990 hsa-miR-3190-3p MIMAT0015074UGGAAGGUagacggccagagag 991 hsa-miR-3191 MIMAT0015075UGGGGACGuagcuggccagacag 992 hsa-miR-3192 MIMAT0015076UCUGGGAGguuguagcaguggaa 993 hsa-miR-3193 MIMAT0015077UCCUGCGUaggaucugaggagu 994 hsa-miR-3194 MIMAT0015078GGCCAGCCaccaggagggcug 995 hsa-miR-3195 MIMAT0015079 CGCGCCGGgcccggguu996 hsa-miR-3196 MIMAT0015080 CGGGGCGGcaggggccuc 997 hsa-miR-548xMIMAT0015081 UAAAAACUgcaauuacuuuca 998 hsa-miR-3197 MIMAT0015082GGAGGCGCaggcucggaaaggcg 999 hsa-miR-3198 MIMAT0015083GUGGAGUCcuggggaauggaga 1000 hsa-miR-3199 MIMAT0015084AGGGACUGccuuaggagaaaguu 1001 hsa-miR-3200 MIMAT0015085CACCUUGCgcuacucaggucug 1002 hsa-miR-3201 MIMAT0015086 GGGAUAUGaagaaaaau1003 hsa-miR-514b-5p MIMAT0015087 UUCUCAAGagggaggcaaucau 1004hsa-miR-514b-3p MIMAT0015088 AUUGACACcucugugagugga 1005 hsa-miR-3202MIMAT0015089 UGGAAGGGagaagagcuuuaau 1006 hsa-miR-1273d MIMAT0015090GAACCCAUgagguugaggcugcagu 1007 hsa-miR-4295 MIMAT0016844CAGUGCAAuguuuuccuu 1008 hsa-miR-4296 MIMAT0016845 AUGUGGGCucaggcuca 1009hsa-miR-4297 MIMAT0016846 UGCCUUCCugucugug 1010 hsa-miR-378cMIMAT0016847 ACUGGACUuggagucagaagagugg 1011 hsa-miR-4293 MIMAT0016848CAGCCUGAcaggaacag 1012 hsa-miR-4294 MIMAT0016849 GGGAGUCUacagcaggg 1013hsa-miR-4301 MIMAT0016850 UCCCACUAcuucacuuguga 1014 hsa-miR-4299MIMAT0016851 GCUGGUGAcaugagaggc 1015 hsa-miR-4298 MIMAT0016852CUGGGACAggaggaggaggcag 1016 hsa-miR-4300 MIMAT0016853 UGGGAGCUggacuacuuc1017 hsa-miR-4304 MIMAT0016854 CCGGCAUGuccagggca 1018 hsa-miR-4302MIMAT0016855 CCAGUGUGgcucagcgag 1019 hsa-miR-4303 MIMAT0016856UUCUGAGCugaggacag 1020 hsa-miR-4305 MIMAT0016857 CCUAGACAccuccaguuc 1021hsa-miR-4306 MIMAT0016858 UGGAGAGAaaggcagua 1022 hsa-miR-4309MIMAT0016859 CUGGAGUCuaggauucca 1023 hsa-miR-4307 MIMAT0016860AAUGUUUUuuccuguuucc 1024 hsa-miR-4308 MIMAT0016861 UCCCUGGAguuucuucuu1025 hsa-miR-4310 MIMAT0016862 GCAGCAUUcauguccc 1026 hsa-miR-4311MIMAT0016863 GAAAGAGAgcugagugug 1027 hsa-miR-4312 MIMAT0016864GGCCUUGUuccugucccca 1028 hsa-miR-4313 MIMAT0016865 AGCCCCCUggccccaaaccc1029 hsa-miR-4315 MIMAT0016866 CCGCUUUCugagcuggac 1030 hsa-miR-4316MIMAT0016867 GGUGAGGCuagcuggug 1031 hsa-miR-4314 MIMAT0016868CUCUGGGAaaugggacag 1032 hsa-miR-4318 MIMAT0016869 CACUGUGGguacaugcu 1033hsa-miR-4319 MIMAT0016870 UCCCUGAGcaaagccac 1034 hsa-miR-4320MIMAT0016871 GGGAUUCUguagcuuccu 1035 hsa-miR-4317 MIMAT0016872ACAUUGCCagggaguuu 1036 hsa-miR-4322 MIMAT0016873 CUGUGGGCucagcgcgugggg1037 hsa-miR-4321 MIMAT0016874 UUAGCGGUggaccgcccugcg 1038 hsa-miR-4323MIMAT0016875 CAGCCCCAcagccucaga 1039 hsa-miR-4324 MIMAT0016876CCCUGAGAcccuaaccuuaa 1040 hsa-miR-4256 MIMAT0016877 AUCUGACCugaugaaggu1041 hsa-miR-4257 MIMAT0016878 CCAGAGGUggggacugag 1042 hsa-miR-4258MIMAT0016879 CCCCGCCAccgccuugg 1043 hsa-miR-4259 MIMAT0016880CAGUUGGGucuaggggucagga 1044 hsa-miR-4260 MIMAT0016881CUUGGGGCauggaguccca 1045 hsa-miR-4253 MIMAT0016882 AGGGCAUGuccagggggu1046 hsa-miR-4251 MIMAT0016883 CCUGAGAAaagggccaa 1047 hsa-miR-4254MIMAT0016884 GCCUGGAGcuacuccaccaucuc 1048 hsa-miR-4255 MIMAT0016885CAGUGUUCagagaugga 1049 hsa-miR-4252 MIMAT0016886 GGCCACUGagucagcacca1050 hsa-miR-4325 MIMAT0016887 UUGCACUUgucucaguga 1051 hsa-miR-4326MIMAT0016888 UGUUCCUCugucucccagac 1052 hsa-miR-4327 MIMAT0016889GGCUUGCAugggggacugg 1053 hsa-miR-4261 MIMAT0016890 AGGAAACAgggaccca 1054hsa-miR-4265 MIMAT0016891 CUGUGGGCucagcucuggg 1055 hsa-miR-4266MIMAT0016892 CUAGGAGGccuuggcc 1056 hsa-miR-4267 MIMAT0016893UCCAGCUCgguggcac 1057 hsa-miR-4262 MIMAT0016894 GACAUUCAgacuaccug 1058hsa-miR-2355 MIMAT0016895 AUCCCCAGauacaauggacaa 1059 hsa-miR-4268MIMAT0016896 GGCUCCUCcucucaggaugug 1060 hsa-miR-4269 MIMAT0016897GCAGGCACagacagcccuggc 1061 hsa-miR-4263 MIMAT0016898 AUUCUAAGugccuuggcc1062 hsa-miR-4264 MIMAT0016899 ACUCAGUCauggucauu 1063 hsa-miR-4270MIMAT0016900 UCAGGGAGucaggggagggc 1064 hsa-miR-4271 MIMAT0016901GGGGGAAGaaaaggugggg 1065 hsa-miR-4272 MIMAT0016902 CAUUCAACuagugauugu1066 hsa-miR-4273 MIMAT0016903 GUGUUCUCugauggacag 1067 hsa-miR-4276MIMAT0016904 CUCAGUGAcucaugugc 1068 hsa-miR-4275 MIMAT0016905CCAAUUACcacuucuuu 1069 hsa-miR-4274 MIMAT0016906 CAGCAGUCccucccccug 1070hsa-miR-4281 MIMAT0016907 GGGUCCCGgggagggggg 1071 hsa-miR-4277MIMAT0016908 GCAGUUCUgagcacaguacac 1072 hsa-miR-4279 MIMAT0016909CUCUCCUCccggcuuc 1073 hsa-miR-4278 MIMAT0016910 CUAGGGGGuuugcccuug 1074hsa-miR-4280 MIMAT0016911 GAGUGUAGuucugagcagagc 1075 hsa-miR-4282MIMAT0016912 UAAAAUUUgcauccagga 1076 hsa-miR-4285 MIMAT0016913GCGGCGAGuccgacucau 1077 hsa-miR-4283 MIMAT0016914 UGGGGCUCagcgaguuu 1078hsa-miR-4284 MIMAT0016915 GGGCUCACaucaccccau 1079 hsa-miR-4286MIMAT0016916 ACCCCACUccugguacc 1080 hsa-miR-4287 MIMAT0016917UCUCCCUUgagggcacuuu 1081 hsa-miR-4288 MIMAT0016918 UUGUCUGCugaguuucc1082 hsa-miR-4292 MIMAT0016919 CCCCUGGGccggccuugg 1083 hsa-miR-4289MIMAT0016920 GCAUUGUGcagggcuauca 1084 hsa-miR-4290 MIMAT0016921UGCCCUCCuuucuucccuc 1085 hsa-miR-4291 MIMAT0016922 UUCAGCAGgaacagcu 1086hsa-miR-4329 MIMAT0016923 CCUGAGACccuaguuccac 1087 hsa-miR-4330MIMAT0016924 CCUCAGAUcagagccuugc 1088 hsa-miR-500b MIMAT0016925AAUCCUUGcuaccugggu 1089 hsa-miR-4328 MIMAT0016926 CCAGUUUUcccaggauu 1090

Example 1 Inhibition of VAMP3 Expression by Single Stranded miR-124Analogs

RT-qPCR Assays—

HCT-116 cells were cultured in McCoy's 5A Medium (Mediatech Inc.)supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.These cells were plated in 96-well culture plates at a density of 6000cells/well 24 hours prior to transfection.

Transfection was carried out using Opti-MEM I Reduced Serum Media(Gibco) and Lipofectamine RNAiMax (Invitrogen) with a final miRNAconcentration of 10 nM for the data in FIGS. 1 and 2, and ranging from30 nM down to 0.01 nM along a 12-point titration curve for the data inFIG. 3.

24 hours after transfection, cells were washed with phosphate-bufferedsaline and processed using the TaqMan® Gene Expression Cells-to-CT™ Kit(Applied Biosystems/Ambion) to extract RNA, synthesize cDNA, and performRT-qPCR using a VAMP3-specific probe (Applied Biosystems) on an ABIPrism 7900HT Sequence Detector.

Reverse transcription conditions were as follows: 60 minutes at 37° C.,followed by 5 minutes at 95° C. RT-qPCR conditions were as follows: 2minutes at 50° C., 10 minutes at 95° C., followed by 40 cycles of 15seconds at 95° C. and 1 minute at 60° C. GUSB mRNA levels were used fordata normalization. Knockdown of VAMP3 was calculated as the two-foldchange in VAMP3 cDNA measured in experimentally-treated cells relativeto the VAMP3 cDNA measured in non-targeting control-treated cells.

Reporter Assays—

HCT-116 cells were cultured in McCoy's 5A Medium (Mediatech Inc.)supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.These cells were plated in 96-well culture plates at a density of 25,000cells/well 24 hours prior to transfection.

Transfection was carried out using Opti-MEM I Reduced Serum Media(Gibco) and Lipofectamine 2000 (Invitrogen) with a final miRNAconcentration of 10 nM for the data in FIGS. 4 and 5, and ranging from30 nM down to 0.01 nM along a 6-point titration curve for the data inFIG. 6. miRNAs were co-transfected with siCHECK2 vectors (Genscript)containing cloned target inserts consisting of a tandem repeat of a seedmatch to miR-124 (2×; FIG. 6A) or a full-length match to miR-124 (2×FL;FIG. 6B).

Twenty-four hours after transfection, transfection medium was replacedwith fresh growth medium. Forty-eight hours after transfection, cellswere lysed and both Firefly- and Renilla-Luciferase activity weremeasured using the Dual-Glo™ Luciferase Assay System (Promega) on aWallac EnVision 2103 Multilabel Reader (PerkinElmer). Firefly-Luciferaseactivity was used to normalize Renilla-Luciferase activity, and thefinal data was calculated as two-fold change of the Renilla-Luciferasesignal in experimentally-treated cells relative to non-targetingcontrol-treated cells.

Oligonucleotide Synthesis—

Oligonucleotides were synthesized using protocols well known in the art(solid phase synthesis) using commercially available phosphoramidites,then purified by reversed phase solid phase extraction (SPE). The C3(C₃₃H₄₃N₂O₅P) and C6 (C₃₆H₄₉N₂O₅P) phosphoramidites were purchased fromChemGenes.

Briefly, the single strand oligonucleotides were synthesized usingphosphoramidite chemistry on an automated solid-phase synthesizer, usingprocedures as are generally known in the art (see, for example, U.S.application Ser. No. 12/064,014, published as US 20090176725). Asynthesis column was packed with solid support derivatized with thefirst nucleoside residue (natural or chemically modified). Synthesis wasinitiated by detritylation of the acid labile 5′-O-dimethoxytrityl groupto release the 5′-hydroxyl. A suitably protected phosphoramidite and asuitable activator in acetonitrile were delivered simultaneously to thesynthesis column resulting in coupling of the amidite to the5′-hydroxyl. The column was then washed with a solvent, such asacetonitrile. An oxidizing solution, such as an iodine solution waspumped through the column to oxidize the phosphite triester linkageP(III) to its phosphotriester P(V) analog. Unreacted 5′-hydroxyl groupswere capped using reagents such as acetic anhydride in the presence of2,6-lutidine and N-methylimidazole. The elongation cycle was resumedwith the detritylation step for the next phosphoramidite incorporation.This process was repeated until the desired sequence was synthesized.The synthesis concluded with the final 5′-terminus protecting group(trityl or 5′-O-dimethoxytrityl).

Upon completion of the synthesis, the solid-support and associatedoligonucleotide were dried under argon pressure or vacuum. Aqueous basewas added and the mixture was heated to effect cleavage of the succinyllinkage, removal of the cyanoethyl phosphate protecting group, anddeprotection of the exocyclic amine protection.

The following process was performed on single strands that do notcontain ribonucleotides. After treating the solid support with theaqueous base, the mixture was filtered to separate the solid supportfrom the deprotected crude synthesis material. The solid support wasthen rinsed with DMSO, which is combined with the filtrate. Theresultant basic solution allows for retention of the5′-O-dimethoxytrityl group to remain on the 5′ terminal position(trityl-on).

For single strands that contain ribonucleotides, the following processwas performed. After treating the solid support with the aqueous base,the mixture was filtered to separate the solid support from thedeprotected crude synthesis material. The solid support was then rinsedwith dimethylsulfoxide (DMSO), which was combined with the filtrate.Fluoride reagent, such as triethylamine trihydrofluoride, was added tothe mixture, and the solution was heated. The reaction was quenched withsuitable buffer to provide a solution of crude single strand with the5′-O-dimethoxytrityl group on the final 5′ terminal position.

The trityl-on solution of each crude single strand was purified usingchromatographic purification, such as SPE RPC purification. Thehydrophobic nature of the trityl group permits stronger retention of thedesired full-length oligo than the non-tritylated truncated failuresequences. The failure sequences were selectively washed from the resinwith a suitable solvent, such as low percent acetonitrile. Retainedoligonucleotides were then detritylated on-column with trifluoroaceticacid to remove the acid-labile trityl group. Residual acid was washedfrom the column, a salt exchange was performed, and a final desalting ofthe material commenced. The full-length oligo was recovered in apurified form with an aqueous-organic solvent. The final product wasthen analyzed for purity (HPLC), identity (Maldi-TOF MS), and yield (UVA₂₆₀). The oligos were dried via lyophilization or vacuum condensation.

Results—

The ability of single-stranded miR-124 analogs to inhibit expression ofa known target, VAMP3, was tested, wherein the miR-124 analogs compriseeither a C3 spacer substituted for one nucleotide, or a C6 spacersubstituted for two nucleotides, at various positions along the strand.

The passenger strand sequence of the miR-124 used in this study is5′-GCAUUCACCGCGUGCCUUAAAU-3′ (SEQ ID NO: 1091), and the guide strandsequence is 5′-UUAAGGCACGCGGUGAAUGCCA-3′ (SEQ ID NO: 1092). The miR-124analogs tested, as well as control molecules, are described in Table 2and below.

TABLE 2 Name Sequence (5′ → 3′) * GIP(guide) UUAAGGCACGCGGUGAAUGCCA (SEQ ID NO: 1092)(passenger) GCAUUCACCGCGUGCCUUAAAU (SEQ ID NO: 1091) 21-8p-c3spacer-20(guide) UAAGGCACGCGGUGAAUGC(C3-spacer)A (SEQ ID NO: 1093)21-8p-c3spacer-19(guide) UAAGGCACGCGGUGAAUG(C3-spacer)CA (SEQ ID NO: 1094) (FIG. 1, 3)c3pos19spacer (FIG. 2) c3pos19 (FIG. 4) 21-8p-c3spacer-18(guide) UAAGGCACGCGGUGAAU(C3-spacer)C CA (SEQ ID NO: 1095)21-8p-c3spacer-17 (guide) UAAGGCACGCGGUGAA(C3-spacer)GCCA (SEQ ID NO: 1096) 21-8p-c3spacer-16(guide) UAAGGCACGCGGUGA(C3-spacer)UGC CA (SEQ ID NO: 1097)21-8p-c3spacer-15 (guide) UAAGGCACGCGGUG(C3-spacer)AUGCCA (SEQ ID NO: 1098) (FIG. 1, 3) c3pos15spacer (FIG. 2)21-8p-c3spacer-14 (guide) UAAGGCACGCGGU(C3-spacer)AAUGCCA (SEQ ID NO: 1099) (FIG. 1, 3) c3pos14 (FIG. 4) 21-8p-c3spacer-13(guide) UAAGGCACGCGG(C3-spacer)GAAUGC CA (SEQ ID NO: 1100)21-8p-c3spacer-12 (guide) UAAGGCACGCG(C3-spacer)UGAAUGCCA (SEQ ID NO: 1101) 21-8p-c3spacer-11(guide) UAAGGCACGC(C3-spacer)GUGAAUGC CA (SEQ ID NO: 1102)21-8p-c3spacer-10 (guide) UAAGGCACG(C3-spacer)GGUGAAUGCCA (SEQ ID NO: 1103) 21-8p-c3spacer-9(guide) UAAGGCAC(C3-spacer)CGGUGAAUGC CA (SEQ ID NO: 1104)21-8p-c3spacer-8 (guide) UAAGGCA(C3-spacer)GCGGUGAAUGCCA (SEQ ID NO: 1105) 21-8p-c3spacer-7(guide) UAAGGC(C3-spacer)CGCGGUGAAUGC CA (SEQ ID NO: 1106)21-8p-c3spacer-6 (guide) UAAGG(C3-spacer)ACGCGGUGAAUGCCA (SEQ ID NO: 1107) 21-8p-c3spacer-5(guide) UAAG(C3-spacer)CACGCGGUGAAUGC CA (SEQ ID NO: 1108)21-8p-c3spacer-4 (guide) UAA(C3-spacer)GCACGCGGUGAAUGCCA (SEQ ID NO: 1109) 21-8p-c3spacer-3(guide) UA(C3-spacer)GGCACGCGGUGAAUGC CA (SEQ ID NO: 1110)21-8p-c3spacer-2 (guide) U(C3-spacer)AGGCACGCGGUGAAUGCCA (SEQ ID NO: 1111) 21-8p-c6spacerdel2-19(guide) UAAGGCACGCGGUGAAUG(C6-spacer)A (SEQ ID NO: 1112)21-8p-c6spacerdel2-18(guide) UAAGGCACGCGGUGAAU(C6-spacer)CA (SEQ ID NO: 1113)21-8p-c6spacerdel2-17 (guide) UAAGGCACGCGGUGAA(C6-spacer)CCA (SEQ ID NO: 1114) 21-8p-c6spacerdel2-16(guide) UAAGGCACGCGGUGA(C6-spacer)GC CA (SEQ ID NO: 1115)21-8p-c6spacerdel2-15 (guide) UAAGGCACGCGGUG(C6-spacer)UGCCA (SEQ ID NO: 1116) (FIG. 1, 3) c6del2po s15 spacer (FIG. 2)21-8p-c6spacerdel2-14 (guide) UAAGGCACGCGGU(C6-spacer)AUGCCA (SEQ ID NO: 1117) 21-8p-c6spacerdel2-13(guide) UAAGGCACGCGG(C6-spacer)AAUGC CA (SEQ ID NO: 1118)21-8p-c6spacerdel2-12 (guide) UAAGGCACGCG(C6-spacer)GAAUGCCA (SEQ ID NO: 1119) 21-8p-c6 spacerdel2-11(guide) UAAGGCACGC(C6-spacer)UGAAUGC CA (SEQ ID NO: 1120)21-8p-c6spacerdel2-10 (guide) UAAGGCACG(C6-spacer)GUGAAUGCCA (SEQ ID NO: 1121) 21-8p-c6spacerdel2-9(guide) UAAGGCAC(C6-spacer)GGUGAAUGC CA (SEQ ID NO: 1122)21-8p-c6spacerdel2-8 (guide) UAAGGCA(C6-spacer)CGGUGAAUGCCA (SEQ ID NO: 1123) 21-8p-c6spacerdel2-7(guide) UAAGGC(C6-spacer)GCGGUGAAUGC CA (SEQ ID NO: 1124) UC3(passenger) B gUaUgaCCgaCUaCgCgUatt B  (SEQ ID NO: 1125) (guide) UACG CG U AG UC GG UC A U A C UU (SEQ ID NO: 1126) miR-124 (FIG. 1)(guide) UUAAGGCACGCGGUGAAUGCCA (SEQ ID NO: 1127) pG (FIG. 2)124(21)-8p-16rrr (guide) UAAGGCACGCGGUGAAUGC CA (SEQ ID NO: 1128)124(21)-8p (FIG. 1) (guide) UAAGGCACGCGGUGAAUGC CA (SEQ ID NO: 1129)G-all-Fluoro (FIG. 2) pG-Fluoro (FIG. 4) GAPDH (guide) AAGUU G UC A UGGA U GA CCU UU (SEQ ID NO: 1131)(passenger) B aggUCaUCCaUgaCaaCUUtt B (SEQ ID NO: 1130) Renilla(guide) UAGUUGCGGACAAUCUGGAtt (SEQ ID NO: 1133)(passenger) UCCAGAUUGUCCGCAACUAtt (SEQ ID NO: 1132) C6delpos15spacer/P(guide) UAAGGCACGCGGUG(C6-spacer)UGC CA (SEQ ID NO: 1116)(passenger) GCAUUCACCGCGUGCCUUAAAU (SEQ ID NO: 1091) pG-Fluoro/Pshort(guide) UAAGGCACGCGGUGAAUGC CA (SEQ ID NO: 1129)(passenger) GCAUUCACCGCGUGCCUUAAU (SEQ ID NO: 1134) * A, U, C, and G =2′-deoxy-2′-fluoro A, U, C, and G A, U, C, and G = 2′-O-methyl (2′-OMe)A, U, C, and G a, g, c and u = deoxy A, U, C,and G t = thymidine A, C,G, and U = ribose A, C, G or U B = inverted abasic

All of the single-stranded molecules in Table 2 contain a 5′ phosphatecap.

“G/P” represents double-stranded miR-124, wherein the duplex has twonucleotide overhangs on the 3′ ends of the passenger and guide strands.The guide strand of the G/P duplex is 22 nucleotides in length.

SEQ ID NOs: 1093-1124 represent analogs of the single-stranded miR-124guide strand. Each of these molecules are a 21-nucleotide version of themiR-124 guide strand that is present in the G/P duplex, missing the5′-uracil nucleotide that is present in the 22-nucleotide G/P miR-124guide strand. All of the nucleotides in these 21-mer analogs, with theexception of the 3′ adenosine, and the adjacent cytosine (if present),are chemically modified on the ribose moiety with 2′-fluoro (depicted asitalicized nucleotides in Table 2). The 3′ adenosine, and the adjacentcytosine (if present), are chemically modified on the ribose moiety with2′-O-methyl (depicted as underlined nucleotides in Table 2). Finally,the 21-mer analogs of the miR-124 guide strand contain either aC3-spacer substituted for one nucleotide (the “c3spacer” analogs) or aC6-spacer substituted for two nucleotides (the “c6spacerdel2” analogs)at the various denoted positions along the strand. For example“21-8p-c3spacer-20” represents a 21-mer miR-124 guide strand analogcontaining an ethylene glycol spacer in the place of the nucleotide atposition 20 within the 21-nucleotide miR-124 guide strand, linking thenucleotides at position 19 and position 21. As another example, theanalog labeled “21-8p-c6spacerdel2-19” represents a 21-mer miR-124 guidestrand analog containing a hexane spacer in the place of the nucleotidesat positions 19 and 20 within the 21-nucleotide miR-124 guide strand,linking the nucleotides at positions 18 and 21. Some of the21-nucleotide miR-124 guide strand analogs have different names inaccompanying Figures, as noted in Table 2. For example, the 21-mermiR-124 guide strand analog represented by SEQ ID NO: 1116 is called“21-8p-c6spacerdel2-15” in FIGS. 1 and 3 and “c6del2pos15spacer” in FIG.2.

“UC3” represents a non-targeting, chemically-modified duplex.

“124(21)-8p-16rrr” represents an analog of the 21-nucleotide version ofthe miR-124 guide strand. This molecule does not contain an internalspacer. All of the nucleotides are modified with 2′-fluoro, with theexception of nucleotides 16-18, which are RNA, and nucleotides 21 and22, which are modified with 2′-O-methyl.

“124(21)-8p” represents a 21-nucleotide version of the miR-124 guide,wherein nucleotides 1-20 are modified with 2′-fluoro and nucleotides 20and 21 are modified with 2′-O-methyl. “124(21)-8p” is the name of thisanalog in FIG. 1; “G-all Fluoro” is the name of this analog in FIG. 2;and, “pG-Fluoro” is the name of this analog in FIG. 4.

“miR-124” is the single-stranded guide strand of the G/P duplex. It is22 nucleotides in length and unmodified.

“C6delpos15spacer/P” represents a double-stranded miR-124 duplex,wherein the guide strand has the structure of “21-8p-c6spacerdel2-15”(SEQ ID NO: 1116), and the passenger strand is the 22-nucleotide miR-124passenger strand (SEQ ID NO: 1091).

FIGS. 1 and 2 show the degree of inhibition of VAMP3 target expressionby the single-stranded miR-124 analogs described in Table 2 using theRT-qPCR assay described above. FIG. 1 shows the degree of inhibition bythe single-stranded miR-124 analogs containing a C3 spacer. FIG. 2 showsthe degree of inhibition by the single-stranded miR-124 analogscontaining a C6 spacer. The longer bars in each figure indicate greaterknockdown of VAMP3. The duplicate bars indicate biological replicates,each representing data from a separate well of cells (on two separateplates) transfected with the indicated nucleic acid molecules. Thespacer appears to be most well-tolerated at position 19, and in thevicinity of position 15, of the miR-124 analogs.

The graph in FIG. 3 depicts the dose-dependent response of VAMP3expression to a subset of the analogs tested in FIGS. 1 and 2 (see Table2 for sequences). VAMP3 expression is depicted along the y-axis, thusdata points with lower values along this axis indicate greater VAMP3expression knockdown. The dose of the miR-124 analog tested is depictedalong the x-axis, ranging from the lowest doses on the left to thehighest doses on the right. Although the double stranded versions (G/Pand c6del2pos15spacer/P) are more potent than the single-strandedanalogs, it is worth noting that the single-stranded “G-all Fluoro”analog (no internal spacer, nucleotides 1-20 are 2′-fluoro, nucleotides20 and 21 are 2′-O-methyl) behaves almost identically to comparablesingle-stranded analogs with a C3 spacer replacing position 15(c3pos15spacer) or position 19 (c3pos19spacer).

FIGS. 4 and 5 show data from a screen of the same single-strandedmiR-124 analogs tested in FIGS. 1 and 2, measuring knockdown of aco-transfected luciferase reporter that carries two target sitesmatching the seed region of miR-124. Thus, the data from this assay is arepresentation of the miRNA activity of the tested analogs. FIG. 4 showsthe degree of inhibition by the single-stranded miR-124 analogs thatcontain a C3 spacer. FIG. 5 shows the degree of inhibition by thesingle-stranded miR-124 analogs that contain a C6 spacer. The duplicatebars indicate biological replicates, each representing data from aseparate well of cells (on two separate plates) transfected with theindicated molecules. Again, the longer bars indicate greater inhibition,showing that the analogs that contain a spacer at position 19, or in thevicinity of position 15, have the greatest knockdown activity.

The graphs in FIG. 6 depict the dose-dependent response of targetexpression inhibition of two different luciferase reporters to a subsetof the analogs tested in FIG. 3. In FIG. 6A, the inhibition activityshown is against a luciferase reporter with two matches to the miR-124seed region. Thus, this is a representation of the miRNA activity of thetested analogs. In FIG. 6B, the inhibition activity shown is against aluciferase reporter with two full-length matches to miR-124 and, thus,represents the siRNA activity of the tested analogs. In both A and B,the G/P curves represent activity by a miR-124 duplex made up entirelyof RNA. The pG-Fluoro/Pshort curves represent activity by a guide strandthat is predominantly modified with 2′-fluoro nucleotides duplexed to anall-RNA passenger strand. The pG-Fluoro curves represent the activity ofa single-stranded miR-124 guide strand analog that is predominantlymodified with 2′-fluoro nucleotides. The c3pos14 and c3pos19 curvesrepresent the activity of analogs of pG-Fluoro, only differing frompG-Fluoro by containing a 3-carbon spacer (C3-spacer) substituted forposition 14 (“c3pos14”) or position 19 (“c3pos19”). All of thesingle-stranded analogs show less potency than either duplexes againstthe reporter with only seed matches, but they are effectively equivalentto each other across all concentrations and show similar activity to theduplexes at the highest concentration (FIG. 6A). Against the reporterwith full-length matches, the all-RNA duplex (“G/P”) was still thestrongest performer, but the spacer-containing single-stranded analogshad activity as strong as or stronger than the duplex with the 2′-fluoroguide strand (“pG-Fluoro/Pshort”) and the single-strand (“pG-Fluoro”).

Example 2 Single-Strand RNAi Knockdown of ApoB mRNA

RT-qPCR Assays (Primary Screens and Dose-Response Curves)—

Hepa1-6 cells were cultured in Dulbecco's Modified Eagle Mediumsupplemented with 10% fetal bovine serum, 1% penicillin-steptomycin, and1% sodium bicarbonate. These cells were plated in a 96-well cultureplates at a density of 3000 cells/well 24 hours prior to transfection.

Transfections were performed using Opti-MEM I Reduced Serum Media andLipofectamine RNAiMAX per the manufacturer's directions. Finalsingle-stranded siRNA concentrations were 100 nM and 10 nM.

Twenty-four hours post-transfection, cells were washed withphosphate-buffered saline and processed using the TaqMan Gene ExpressionCells-to-CT™ Kit, per manufacturer's instructions, to extract RNA,synthesize cDNA, and perform RT-qPCR using an ApoB specific Taqmanprimer/probe set on an ABI Prism 7900HT Sequence Detector.

Reverse transcription conditions were as follows: 60 minutes at 37° C.followed by 5 minutes at 95° C. RT-qPCR conditions were as follows: 2minutes at 50° C., 10 minutes at 95° C., followed by 40 cycles of 15seconds at 95° C. and 1 minute at 60° C. GADPH mRNA levels were used fordata normalization.

Knockdown of ApoB was calculated as the two-fold change in ApoB cDNAmeasured in experimentally-treated cells relative to the ApoB cDNAmeasured in non-targeting, control-treated cells.

Results—

The knockdown of ApoB mRNA was measured using single strand (guidestrand) oligonucleotides with a C3 spacer incorporated at eitherposition 15, 16, 17, 18, or 19 (relative to the 5′ of the oligo) at twodifferent concentrations (100 nM and 10 nM). The results are shown inFIG. 7. All of the single strand molecules tested are composed of2′-deoxy-2′-fluoro nucleotides at both pyrimidine and purine nucleotidesand a 5′ phosphate. The two 3′ terminal nucleotides of each molecule are2′-O-methyl nucleotides. Single strand molecule “485” (see FIG. 7;5′-UUAAGAGAAGCCUUACUGGUU-3′ (SEQ ID NO: 1135)) is a 21-nucleotidemolecule that does not contain a C3 spacer and targets ApoB mRNA atnucleotide position 485. A C3 spacer is incorporated into molecule 485at positions 15 (“485 c3@pos15”; SEQ ID NO: 1136), 16 (“485 c3@pos16”;SEQ ID NO: 1137), 17 (“485 c3@pos17”; SEQ ID NO: 1138), 18 (“485c3@pos18”; SEQ ID NO: 1139) or 19 (“485 c3@pos19”; SEQ ID NO: 1140)(i.e., the spacer takes the place of the indicated nucleotide of the 485molecule). For example, signal strand molecule “485 c3@pos15” isrepresented by: 5′-UUAAGAGAAGCCUU(C3-spacer)CUGGUU-3′; SEQ ID NO: 1136).As shown in FIG. 7, inclusion of the C3 spacer at positions 18 or 19 isboth well tolerated and improves mRNA knockdown at two differentconcentrations (100 nM and 10 nM). This data indicates thatincorporation of a non-nucleotide C3 carbon spacer at the 3′ end of asingle strand RNA interference oligonucleotide improves the potency ofmRNA knockdown (FIG. 7).

To evaluate whether the incorporation of a C3 spacer in single strandswas more broadly applicable, 30 different single strand sequencestargeting ApoB, each with a C3 spacer at either position 18 (FIG. 8) orposition 19 (FIG. 9), were evaluated at two concentrations (100 nM and10 nM). In FIGS. 8 and 9, the single-stranded molecules are notated bythe position within the ApoB mRNA which they target. All of thesingle-stranded molecules tested are composed of 2′-deoxy-2′-fluoronucleotides at both pyrimidine and purine nucleotides and a 5′phosphate. The two 3′ terminal nucleotides of each molecule are2′-O-methyl nucleotides. The sequence identifiers (SEQ ID NOs:) of thesingle stranded RNAi molecules evaluated in FIGS. 8 and 9 are listed inTable 3.

TABLE 3 ApoB Target C3-position 18 C3-position 19 No spacer Region(FIGS. 8 and 10) (FIGS. 9 and 10) (FIG. 10) 19 SEQ ID NO: 1141 SEQ IDNO: 1170 SEQ ID NO: 1199 248 SEQ ID NO: 1142 SEQ ID NO: 1171 SEQ ID NO:1200 397 SEQ ID NO: 1143 SEQ ID NO: 1172 SEQ ID NO: 1201 485 SEQ ID NO:1139 SEQ ID NO: 1140 SEQ ID NO: 1135 601 SEQ ID NO: 1144 SEQ ID NO: 1173SEQ ID NO: 1202 719 SEQ ID NO: 1145 SEQ ID NO: 1174 SEQ ID NO: 1203 780SEQ ID NO: 1146 SEQ ID NO: 1175 SEQ ID NO: 1204 1124 SEQ ID NO: 1147 SEQID NO: 1176 SEQ ID NO: 1205 1445 SEQ ID NO: 1148 SEQ ID NO: 1177 SEQ IDNO: 1206 1446 SEQ ID NO: 1149 SEQ ID NO: 1178 SEQ ID NO: 1207 1611 SEQID NO: 1150 SEQ ID NO: 1179 SEQ ID NO: 1208 1983 SEQ ID NO: 1151 SEQ IDNO: 1180 SEQ ID NO: 1209 3214 SEQ ID NO: 1152 SEQ ID NO: 1181 SEQ ID NO:1210 3614 SEQ ID NO: 1153 SEQ ID NO: 1182 SEQ ID NO: 1211 4542 SEQ IDNO: 1154 SEQ ID NO: 1183 SEQ ID NO: 1212 6548 SEQ ID NO: 1155 SEQ ID NO:1184 SEQ ID NO: 1213 6930 SEQ ID NO: 1156 SEQ ID NO: 1185 SEQ ID NO:1214 6981 SEQ ID NO: 1157 SEQ ID NO: 1186 SEQ ID NO: 1215 7044 SEQ IDNO: 1158 SEQ ID NO: 1187 SEQ ID NO: 1216 9414 SEQ ID NO: 1159 SEQ ID NO:1188 SEQ ID NO: 1217 9514 SEQ ID NO: 1160 SEQ ID NO: 1189 SEQ ID NO:1218 9621 SEQ ID NO: 1161 SEQ ID NO: 1190 SEQ ID NO: 1219 10162 SEQ IDNO: 1162 SEQ ID NO: 1191 SEQ ID NO: 1220 10167 SEQ ID NO: 1163 SEQ IDNO: 1192 SEQ ID NO: 1221 10168 SEQ ID NO: 1164 SEQ ID NO: 1193 SEQ IDNO: 1222 10219 SEQ ID NO: 1165 SEQ ID NO: 1194 SEQ ID NO: 1223 10455 SEQID NO: 1166 SEQ ID NO: 1195 SEQ ID NO: 1224 10517 SEQ ID NO: 1167 SEQ IDNO: 1196 SEQ ID NO: 1225 12673 SEQ ID NO: 1168 SEQ ID NO: 1197 SEQ IDNO: 1226 13666 SEQ ID NO: 1169 SEQ ID NO: 1198 SEQ ID NO: 1227

In FIGS. 8 and 9, the data was normalized to the correspondingsingle-stranded molecule without the C3 spacer. Knockdown amounts thatare equivalent to the single-stranded controls (without C3 spacer) wouldbe centered at 0, while positive values indicate that incorporation ofthe C3 spacer confers an improvement in mRNA knockdown. Negative valuesindicate a deleterious effect of C3 spacer inclusion. Note that due toexperimental variation with in vitro assays, only values greater than+0.5 or less than −0.5 are considered significant. For example,inclusion of the C3 spacer in ApoB molecule 485 at position 18 does nothave a significant improvement over the single strand control since thedifference in knockdown shown in the FIG. 8 is less than 0.5. However,inclusion of the C3 spacer in the same single strand guide molecule atposition 19 has a significant improvement in knockdown (see FIG. 9).Overall, inclusion of the C3 spacer at position 19 is preferred asincorporation at this position seems to improve the potency of mRNAknockdown for the majority of the 30 different sequences tested (73%).

In FIG. 10, ApoB mRNA knockdown at 100 nM concentration using singlestranded molecules targeting each of the 30 different ApoB target sitestested in FIGS. 8 and 9 were compared—single strands without C3 spacer(“all-flu-p”), with C3 spacer at position 18 (“all-flu-c3-18-p”), andwith C3 spacer at position 19 (“all-flu-c3-19-p”). All of thesingle-stranded molecules tested are composed of 2′-deoxy-2′-fluoronucleotides at both pyrimidine and purine nucleotides and a 5′phosphate. The two 3′ terminal nucleotides of each molecule are2′-O-methyl nucleotides. The sequence identifiers (SEQ ID NOs:) of thesingle stranded RNAi molecules evaluated in FIG. 10 are listed in Table3. FIG. 10 demonstrates the range in overall efficacy of mRNA knockdownfor different single-stranded sequences. For example, single strandmolecules targeting ApoB target site 485 is maximally effective, whileothers like those targeting ApoB target site 780 or 10219 have limitedmRNA knockdown. For each of the 30 different sequences, the mRNAknockdown shown in FIG. 10 was normalized to the corresponding strandswhich do not contain the C3 spacer (“all-flu-p”).

What is claimed:
 1. A single-stranded RNA molecule that mediates RNAinterference against a target RNA, wherein said single-stranded RNAcomprises: (a) a nucleic acid portion comprising a first nucleotideportion (N1) and a second nucleotide portion (N2) that are not selfcomplementary, wherein said nucleic acid portion comprises at least 8nucleotides that can base pair with a target site of the target RNA, andwherein the total number of nucleotides within the nucleic acid portionis from 8 to 26 nucleotides; and, (i) an internal spacer portion,wherein said spacer portion comprises at least a first non-nucleotidespacer portion (S1) that covalently links the first and secondnucleotide portions.
 2. A molecule of claim 1 comprising the followingstructure:5′ N1-S1-N2 3′ wherein: (a) N1 contains either one nucleotide or acontiguous stretch of nucleotides; (b) S1 contains one or morenon-nucleotide spacers covalently linking N1 and N2; and, (c) N2contains either one nucleotide or a contiguous stretch of nucleotides.3. A molecule of claim 1 or 2, wherein S is an aliphatic or aromaticorganic group.
 4. A molecule of any one of claims 1-3, wherein S1 is aC₁-C₁₂ alkyl chain that is optionally substituted.
 5. A molecule ofclaim 4, wherein said alkyl chain is optionally substituted withcholesterol.
 6. A molecule of any one of claims 1-5, wherein S1 isselected from a group consisting of a C3 alkyl, a C6 alkyl, andpolyethylene glycol.
 7. A molecule of any one of claims 1-6, wherein N1is 13 to 20 nucleotides long.
 8. A molecule of any one of claims 1-7,wherein the total number of nucleotides within the nucleic acid portionis about 19 to about 21 nucleotides.
 9. A molecule of any one of claims1-8, wherein the target site is within a untranslated region of thetarget RNA.
 10. A molecule of claim 9, wherein the at least 8nucleotides that can base pair with the target site is the whole or apart of a seed sequence of a naturally-occurring, endogenous miRNAnucleotide sequence.
 11. A molecule of claim 10, wherein S takes theplace of from one to 4 internal nucleotides of the naturally-occurring,endogenous miRNA nucleotide sequence.
 12. A molecule of any one ofclaims 9-11, wherein the nucleic acid portion of the molecule is atleast 50% homologous to the naturally-occurring, endogenous miRNAnucleotide sequence.
 13. A molecule of any one of claims 1-8, whereinthe target site is within a gene coding region of the target RNA.
 14. Amolecule of any one of claims 1-13, wherein the nucleic acid portion ofthe molecule is at least 90% complementary to the target site.
 15. Amolecule of any one of claims 1-14, wherein the nucleic acid portioncomprises at least 20 nucleotides that can base pair with the targetsite.
 16. A molecule of any one of claims 1-15, wherein the nucleic acidportion further comprises a third nucleotide portion (N3) and theinternal spacer portion further comprises a second non-nucleotide spacerportion (S2).
 17. A molecule of any one of claims 1-16, wherein at leastone nucleotide has a modified sugar.
 18. A molecule of any one of claims1-17, wherein at least one nucleotide has a modified internucleosidelinkage.
 19. A molecule of any one of claims 1-18, having a terminal capat the 5′-end, the 3′-end, or both the 5′- and 3′-ends.
 20. Acomposition comprising the single-stranded RNA molecule of any one ofclaims 1-19 and a pharmaceutically acceptable carrier.
 21. A compositionof claim 20, further comprising a liposome, a hydrogel, a cyclodextrin,a biodegradable nanocapsule, a bioadhesive microsphere, or aproteinaceous vector.
 22. A method of reducing the expression of anendogenous RNA target gene in a cell comprising administering acomposition of claim 20 or claim 21.